Pharmaceutical compositions comprising FGF19 variants

ABSTRACT

Provided herein are pharmaceutical compositions, formulations and dosage forms comprising variants of fibroblast growth factor 19 (FGF19) proteins and peptide sequences (and peptidomimetics) and fusions of FGF19 and/or fibroblast growth factor 21 (FGF21) proteins and peptide sequences (and peptidomimetics), and variants of fusions of FGF19 and/or FGF21 proteins and peptide sequences (and peptidomimetics). Methods of using the pharmaceutical compositions, formulations and dosage forms are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 national stage application of internationalapplication Serial No. PCT/US2015/056830 filed Oct. 22, 2015, whichclaims the benefit of priority to U.S. Ser. No. 62/067,824 filed Oct.23, 2014, which is incorporated herein by reference in its entirety.

1. FIELD

Provided herein are pharmaceutical compositions, formulations and dosageforms comprising variants of fibroblast growth factor 19 (FGF19)proteins and peptide sequences (and peptidomimetics) and fusions ofFGF19 and/or fibroblast growth factor 21 (FGF21) proteins and peptidesequences (and peptidomimetics), and variants of fusions of FGF19 and/orFGF21 proteins and peptide sequences (and peptidomimetics). Methods ofusing the pharmaceutical compositions, formulations and dosage forms arealso provided herein.

2. BACKGROUND

Drug substances are usually administered as part of a formulation incombination with one or more other agents that serve varied andspecialized pharmaceutical functions. Dosage forms of various types maybe made through selective use of pharmaceutical excipients. Aspharmaceutical excipients have various functions and contribute to thepharmaceutical formulations in many different ways, e.g.,solubilization, dilution, thickening, stabilization, preservation,coloring, flavoring, etc. The properties that are commonly consideredwhen formulating an active drug substance include bioavailability, easeof manufacture, ease of administration, and stability of the dosageform. Due to the varying properties of active drug substances beingformulated, dosage forms typically require pharmaceutical excipientsthat are uniquely tailored to the active drug substance in order toachieve advantageous physical and pharmaceutical properties.

Provided herein are formulations and dosage forms comprising variants offibroblast growth factor 19 (FGF19) proteins and peptide sequences (andpeptidomimetics) and fusions of FGF19 and/or fibroblast growth factor 21(FGF21) proteins and peptide sequences (and peptidomimetics), andvariants of fusions of FGF19 and/or FGF21 proteins and peptide sequences(and peptidomimetics). Such proteins and peptide sequences are useful intreating, preventing and/or managing various disease or disorders,including, but not limited to, bile acid-related or associateddisorders, hyperglycemic conditions, insulin resistance,hyperinsulinemia, glucose intolerance, metabolic syndrome, and metabolicdiseases, as well as certain cancers. Thus, a need exists as topharmaceutical compositions and dosage forms of such proteins andpeptide sequences having advantageous physical and pharmaceuticalproperties. The present invention satisfies this need and providesrelated benefits.

3. SUMMARY

In one aspect, provided herein a pharmaceutical composition comprising apeptide or peptide sequence, and pharmaceutically acceptable carrier. Incertain embodiments, the pharmaceutically acceptable carrier comprisesTris, trehalose or polysorbate-20 (TWEEN-20), or any combinationthereof.

In some embodiments, the pharmaceutical composition comprises peptideselected from a group consisting of variants of FGF19 peptide sequences,fusions of FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences having one or moreactivities, such as bile acid homeostasis modulating activity. Suchvariants and fusions (chimeras) of FGF19 and/or FGF21 peptide sequencesinclude sequences that are used for treating a bile acid-related orassociated disorder. Such variants and fusions (chimeras) of FGF19and/or FGF21 peptide sequences also include sequences that do notsubstantially or significantly increase or induce hepatocellularcarcinoma (HCC) formation or HCC tumorigenesis. Such variants andfusions (chimeras) of FGF19 and/or FGF21 peptide sequences furtherinclude sequences that do not induce a substantial elevation or increasein lipid profile.

In one embodiment, the peptide is a chimeric peptide comprising: a) anN-terminal region comprising at least seven amino acid residues, theN-terminal region having a first amino acid position and a last aminoacid position, wherein the N-terminal region comprises DSSPL (SEQ IDNO:121) or DASPH (SEQ ID NOL122); and b) a C-terminal region comprisinga portion of SEQ ID NO:99 (FGF19), the C-terminal region having a firstamino acid position and a last amino acid position, wherein theC-terminal region comprises amino acid residues 16-29 of SEQ ID NO:99(FGF19) (WGDPIRLRHLYTSG; SEQ ID NO:169), wherein the W residuecorresponds to the first amino acid position of the C-terminal region,to modulate bile acid homeostasis or treat the bile acid-related orassociated disorder.

In another embodiment, the peptide is a chimeric peptide comprising: a)an N-terminal region comprising a portion of SEQ ID NO:100 (FGF21), theN-terminal region having a first amino acid position and a last aminoacid position, wherein the N-terminal region comprises amino acidresidues GQV, and wherein the V residue corresponds to the last aminoacid position of the N-terminal region; and b) a C-terminal regioncomprising a portion of SEQ ID NO:99 (FGF19), the C-terminal regionhaving a first amino acid position and a last amino acid position,wherein the C-terminal region comprises amino acid residues 21-29 of SEQID NO:99 (FGF19), RLRHLYTSG (SEQ ID NO:185), and wherein the R residuecorresponds to the first position of the C-terminal region, to modulatebile acid homeostasis or treat the bile acid-related or associateddisorder.

In a further embodiment, the peptide is a chimeric peptide comprising:a) an N-terminal region comprising a portion of SEQ ID NO:100 (FGF21),the N-terminal region having a first amino acid position and a lastamino acid position, wherein the N-terminal region comprises at least 5contiguous amino acids of SEQ ID NO:100 (FGF21) including the amino acidresidues GQV, and wherein the V residue corresponds to the last aminoacid position of the N-terminal region; and b) a C-terminal regioncomprising a portion of SEQ ID NO:99 (FGF19), the C-terminal regionhaving a first amino acid position and a last amino acid position,wherein the C-terminal region comprises amino acid residues 21-29 of SEQID NO:99 (FGF19), RLRHLYTSG (SEQ ID NO:185), and wherein the R residuecorresponds to the first position of the C-terminal region, to modulatebile acid homeostasis or treat the bile acid-related or associateddisorder.

In an additional embodiment, the peptide comprises or consists of anyof: a) a FGF19 sequence variant having one or more amino acidsubstitutions, insertions or deletions compared to a reference or wildtype FGF19; b) a FGF21 sequence variant having one or more amino acidsubstitutions, insertions or deletions compared to a reference or wildtype FGF21; c) a portion of a FGF19 sequence fused to a portion of aFGF21 sequence; or d) a portion of a FGF19 sequence fused to a portionof a FGF21 sequence, wherein the FGF19 and/or FGF21 sequence portion(s)have one or more amino acid substitutions, insertions or deletionscompared to a reference or wild type FGF19 and/or FGF21, to modulatebile acid homeostasis or treat the bile acid-related or associateddisorder.

In various particular embodiments, a chimeric peptide sequence has anN-terminal region with at least 6 contiguous amino acids of SEQ IDNO:100 (FGF21) including the amino acid residues GQ; or has anN-terminal region with at least 7 contiguous amino acids of SEQ IDNO:100 (FGF21) including the amino acid residues GQV.

In some embodiments, the peptide comprises i) a FGF19 sequence varianthaving one or more amino acid substitutions, insertions or deletionscompared to a reference or wild type FGF19; ii) a FGF21 sequence varianthaving one or more amino acid substitutions, insertions or deletionscompared to a reference or wild type FGF21; iii) a portion of a FGF19sequence fused to a portion of a FGF21 sequence; or iv) a portion of aFGF19 sequence fused to a portion of a FGF21 sequence, wherein the FGF19and/or FGF21 sequence portion(s) have one or more amino acidsubstitutions, insertions or deletions compared to a reference or wildtype FGF19 and/or FGF21.

In various additional embodiments, a peptide sequence has amino-terminalamino acids 1-16 of SEQ ID NO:100 (FGF21) fused to carboxy-terminalamino acids 21-194 of SEQ ID NO:99 (FGF19), or the peptide sequence hasamino-terminal amino acids 1-147 of SEQ ID NO:99 (FGF19) fused tocarboxy-terminal amino acids 147-181 of SEQ ID NO:100 (FGF21) (M41), orthe peptide sequence has amino-terminal amino acids 1-20 of SEQ ID NO:99(FGF19) fused to carboxy-terminal amino acids 17-181 of SEQ ID NO:100(FGF21) (M44), or the peptide sequence has amino-terminal amino acids1-146 of SEQ ID NO:100 (FGF21) fused to carboxy-terminal amino acids148-194 of SEQ ID NO:99 (FGF19) (M45), or the peptide sequence hasamino-terminal amino acids 1-20 of SEQ ID NO:99 (FGF19) fused tointernal amino acids 17-146 of SEQ ID NO:100 (FGF21) or fused tocarboxy-terminal amino acids 148-194 of SEQ ID NO:99 (FGF19) (M46).

In various further embodiments, a peptide sequence has at least oneamino acid substitution to amino acid residues 125-129 of SEQ ID NO:99(FGF19), EIRPD; at least one amino acid substitution to amino acidresidues 126-128 of SEQ ID NO:99 (FGF19), IRP; or at least one aminoacid substitution to amino acid residues 127-128 of SEQ ID NO:99(FGF19), RP, or at least one amino acid substitution to amino acidresidues 1-124 of SEQ ID NO:99 (FGF19) and/or to amino acid residues130-194 of SEQ ID NO:99 (FGF19). More specifically, for example, apeptide sequence with a substitution to one of amino acid residues127-128 of SEQ ID NO:99 (FGF19), IRP, wherein at least one amino acidsubstitution is R127L or P128E.

Methods and uses provided herein can be practiced using anypharmaceutical composition comprising a peptide or chimeric sequence, asset forth herein. For example, a sequence that includes or consists ofany peptide sequence set forth herein as M1-M98, M101 to M160, or M200to M207, or SEQ ID NOs:1 to 98, or 101 to 135, or 138 to 212. In otherembodiments, the peptide sequence includes or consists of any sequenceset forth in Table 1. In yet other embodiments, the peptide sequencethat includes or consists of any sequence set forth in the SequenceListing herein. In certain embodiments, the amino acid sequence of thepeptide comprises at least one amino acid substitution in the Loop-8region of FGF19, or the corresponding FGF19 sequence thereof in avariant peptide provided herein. In certain embodiments, the amino acidsequence of the peptide comprises one amino acid substitution to theEIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in theLoop-8 region of FGF19. In some embodiments, the amino acid sequence ofthe peptide comprises two amino acid substitutions to the EIRPD (aminoacids 2-6 of SEQ ID NO:190) amino acid sequence in the Loop-8 region ofFGF19. In other embodiments, the amino acid sequence of the peptidecomprises three amino acid substitutions to the EIRPD (amino acids 2-6of SEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19. Incertain embodiments, the amino acid sequence of the peptide comprisesfour amino acid substitutions to the EIRPD (amino acids 2-6 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19. In someembodiments, the amino acid sequence of the peptide comprises five aminoacid substitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190) aminoacid sequence in the Loop-8 region of FGF19. In certain embodiments, theamino acid sequence of the peptide comprises one amino acid substitutionto the IRP (amino acids 3-5 of SEQ ID NO:190) amino acid sequence in theLoop-8 region of FGF19. In some embodiments, the amino acid sequence ofthe peptide comprises two amino acid substitutions to the IRP (aminoacids 3-5 of SEQ ID NO:190) amino acid sequence in the Loop-8 region ofFGF19. In other embodiments, the amino acid sequence of the peptidecomprises three amino acid substitutions to the IRP (amino acids 3-5 ofSEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19. Incertain embodiments, the amino acid sequence of the peptide comprisesone amino acid substitution to the RP (amino acids 4-5 of SEQ ID NO:190)amino acid sequence in the Loop-8 region of FGF19. In some embodiments,the amino acid sequence of the peptide comprises two amino acidsubstitutions to the RP (amino acids 4-5 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In certain embodiments, theamino acid substitution to the RP (amino acids 4-5 of SEQ ID NO:190)amino acid sequence in the Loop-8 region of FGF19 is an Arg (R) to Leu(L) substitution. In other embodiments, the substitution to the RP(amino acids 4-5 of SEQ ID NO:190) amino acid sequence in the Loop-8region of FGF19 is a Pro (P) to Glu (E) substitution. In someembodiments, the substitutions to the RP (amino acids 4-5 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19 is an Arg (R)to Leu (L) substitution and a Pro (P) to Glu (E) substitution. Inspecific embodiments, the foregoing substitution(s) in the Loop-8 regionof FGF19 is in the corresponding FGF19 sequence thereof in a variantpeptide provided herein. That is, said substitutions within acorresponding FGF19 sequence (e.g., EIRPD, IRP or RP) of a peptidevariant provided herein is also contemplated.

Methods and uses provided herein can be practiced using a pharmaceuticalcomposition comprising a peptide or chimeric sequence of any suitablelength. In particular embodiments, the N-terminal or C-terminal regionof the peptide or chimeric sequence is from about 20 to about 200 aminoacid residues in length. In other particular aspects, a peptide orchimeric sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more amino acid deletions from the amino terminus,the carboxy-terminus or internally. In further particular embodiments, apeptide or chimeric sequence has an N-terminal region, or a C-terminalregion that includes or consists of an amino acid sequence of about 5 to10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to80, 80 to 90, 90 to 100 or more amino acids. In additional moreparticular embodiments, a peptide or chimeric sequence has a FGF19sequence portion, or a FGF21 sequence portion that includes or consistsof an amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or moreamino acids of FGF19 or FGF21.

In yet additional embodiments, a peptide sequence or a chimeric peptidesequence has a WGDPI (SEQ ID NO:170) sequence motif corresponding to theWGDPI (SEQ ID NO:170) sequence of amino acids 16-20 of SEQ ID NO:99(FGF19); has a substituted, mutated or absent WGDPI (SEQ ID NO:170)sequence motif corresponding to FGF19 WGDPI (SEQ ID NO:170) sequence ofamino acids 16-20 of FGF19; has a WGDPI (SEQ ID NO:170) sequence withone or more amino acids substituted, mutated or absent. In various otherfurther aspects, the peptide sequence is distinct from a FGF19 variantsequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ IDNO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ IDNO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the FGF19 WGDPI (SEQ ID NO:170) sequence atamino acids 16-20.

In yet further embodiments, a peptide sequence or a chimeric peptidesequence has N-terminal region comprises amino acid residues VHYG (SEQID NO:101), wherein the N-terminal region comprises amino acid residuesDASPHVHYG (SEQ ID NO:102), or the N-terminal region comprises amino acidresidues DSSPLVHYG (SEQ ID NO:103). More particularly, in one aspect theG corresponds to the last position of the N-terminal region.

In various additional aspects, the N-terminal region comprises aminoacid residues DSSPLLQ (SEQ ID NO:104), where the Q residue is the lastamino acid position of the N-terminal region, or comprises amino acidresidues DSSPLLQFGGQV (SEQ ID NO:105), where the V residue correspondsto the last position of the N-terminal region.

In certain embodiments, an N-terminal region comprises or consists of(or further comprises or consists of): RHPIP (SEQ ID NO:106), where R isthe first amino acid position of the N-terminal region; or HPIP (SEQ IDNO:107), where H is the first amino acid position of the N-terminalregion; or RPLAF (SEQ ID NO:108), where R is the first amino acidposition of the N-terminal region; or PLAF (SEQ ID NO:109), where P isthe first amino acid position of the N-terminal region; or R, where R isthe first amino acid position of the N-terminal region.

In various other aspects, a peptide or chimeric sequence has: amino acidresidues HPIP (SEQ ID NO:107), which are the first 4 amino acid residuesof the N-terminal region. In various still further aspects, a peptide orchimeric sequence has: an R residue at the first position of theN-terminal region, or the first position of the N-terminal region is anM residue, or the first and second positions of the N-terminal region isan MR sequence, or the first and second positions of the N-terminalregion is an RM sequence, or the first and second positions of theN-terminal region is an RD sequence, or the first and second positionsof the N-terminal region is an DS sequence, or the first and secondpositions of the N-terminal region is an MD sequence, or the first andsecond positions of the N-terminal region is an MS sequence, or thefirst through third positions of the N-terminal region is an MDSsequence, or the first through third positions of the N-terminal regionis an RDS sequence, or the first through third positions of theN-terminal region is an MSD sequence, or the first through thirdpositions of the N-terminal region is an MSS sequence, or the firstthrough third positions of the N-terminal region is an DSS sequence, orthe first through fourth positions of the N-terminal region is an RDSS(SEQ ID NO:115), sequence, or the first through fourth positions of theN-terminal region is an MDSS (SEQ ID NO:116), sequence, or the firstthrough fifth positions of the N-terminal region is an MRDSS (SEQ IDNO:117), sequence, or the first through fifth positions of theN-terminal region is an MSSPL (SEQ ID NO:113) sequence, or the firstthrough sixth positions of the N-terminal region is an MDSSPL (SEQ IDNO:110) sequence, or the first through seventh positions of theN-terminal region is an MSDSSPL (SEQ ID NO:111) sequence.

In various other particular aspects, a peptide or chimeric sequence hasat the N-terminal region first amino acid position an “M” residue, an“R” residue, a “S” residue, a “H” residue, a “P” residue, a “L” residueor an “D” residue. In various alternative particular aspects, a peptideor chimeric sequence peptide sequence does not have a “M” residue or an“R” residue at the first amino acid position of the N-terminal region.

In further various other embodiments, a peptide or chimeric sequence hasan N-terminal region with any one of the following sequences: MDSSPL(SEQ ID NO:110), MSDSSPL (SEQ ID NO:111), SDSSPL (SEQ ID NO:112), MSSPL(SEQ ID NO:113) or SSPL (SEQ ID NO:114).

In various still additional aspects, a peptide or chimeric sequence hasa residue at the last position of the C-terminal region that correspondsto about residue 194 of SEQ ID NO:99 (FGF19). In still otherembodiments, a peptide sequence or a chimeric peptide sequence anaddition of amino acid residues 30-194 of SEQ ID NO:99 (FGF19) at theC-terminus, resulting in a chimeric polypeptide having at the lastposition of the C-terminal region that corresponds to about residue 194of SEQ ID NO:99 (FGF19). In further other embodiments, a chimericpeptide sequence or peptide sequence comprises all or a portion of aFGF19 sequence (e.g., SEQ ID NO:99), positioned at the C-terminus of thepeptide, or where the amino terminal “R” residue is deleted from thepeptide.

In more particular embodiments, a chimeric peptide sequence or peptidesequence comprises or consists of any of M1-M98 variant peptidesequences, or a subsequence or fragment of any of the M1-M98 variantpeptide sequences. Methods and uses provided herein can also bepracticed using a peptide or chimeric sequence, as set forth herein. Forexample, a sequence that comprises or consists of any peptide sequenceset forth herein as M1 to M98, M101 to M160, or M200 to M207 or SEQ IDNOs:1 to 98, 101 to 135, 138 to 212, or a peptide sequence thatcomprises of consists of any sequence set forth in Table 1, or a peptidesequence that comprises or consists of any sequence set forth in theSequence Listing herein.

In various more particular embodiments, a peptide sequence comprises orconsists of any one of the following sequences:

RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M3) (SEQ ID NO: 3);RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140) (SEQ ID NO: 194);RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160) (SEQ ID NO: 196);RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M69) (SEQ ID NO: 69);RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M52) (SEQ ID NO: 52);RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPS FEK(M5) (SEQ ID NO: 5);HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M5-R) (SEQ ID NO: 160);HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71) (SEQ ID NO: 71);HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72) (SEQ ID NO: 72);HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73) (SEQ ID NO: 73);RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M1) (SEQ ID NO: 1 or 139);RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M2) (SEQ ID NO: 2 or 140);RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M48) (SEQ ID NO: 48 or 6 or 148);RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M49) (SEQ ID NO: 49 or 7 or 149);RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M50) (SEQ ID NO: 50);RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M51) (SEQ ID NO: 51 or 36 or 155);MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M53) (SEQ ID NO: 192);MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70) (SEQ ID NO: 70);RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M139) (SEQ ID NO: 193); orRPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141) (SEQ ID NO: 195);RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160) (SEQ ID NO: 196);or a subsequence or fragment thereof any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the R terminal residue (R residue at the N-terminus) isdeleted.

In other embodiments, the peptide comprises or consists of:

RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M200) (SEQ ID NO: 197);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In some embodiments, the peptide comprises or consists of:

RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M201) (SEQ ID NO: 98);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In certain embodiments, the peptide comprises or consists of:

RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M202) (SEQ ID NO: 199);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In other embodiments, the peptide comprises or consists of:

RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M203) (SEQ ID NO: 200);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In some embodiments, the peptide comprises or consists of:

RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M204) (SEQ ID NO: 201);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In certain embodiments, the peptide comprises or consists of:

RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M205) (SEQ ID NO: 202);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In some embodiments, the peptide comprises or consists of:

RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M206) (SEQ ID NO: 203);or a subsequence or fragment thereof. In one embodiment, the N-terminalR residue is deleted.

In other embodiments, the peptide comprises or consists of:

MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M207) (SEQ ID NO: 204);or a subsequence or fragment thereof.

In some embodiments, the peptide is a variant peptide designated M139.In one embodiment, the peptide comprises an amino acid sequence setforth in SEQ ID NO:193. In other embodiments, the peptide consists of anamino acid sequence set forth in SEQ ID NO:193. In some embodiments, thepeptide is a variant peptide designated M140. In one embodiment, thepeptide comprises an amino acid sequence set forth in SEQ ID NO:194. Inother embodiments, the peptide consists of an amino acid sequence setforth in SEQ ID NO:194. In some embodiments, the peptide is a variantpeptide designated M141. In one embodiment, the peptide comprises anamino acid sequence set forth in SEQ ID NO:195. In other embodiments,the peptide consists of an amino acid sequence set forth in SEQ IDNO:195. In some embodiments, the peptide is a variant peptide designatedM160. In one embodiment, the peptide comprises an amino acid sequenceset forth in SEQ ID NO:196. In other embodiments, the peptide consistsof an amino acid sequence set forth in SEQ ID NO:196. In someembodiments, the peptide is a variant peptide designated M200. In oneembodiment, the peptide comprises an amino acid sequence set forth inSEQ ID NO:197. In other embodiments, the peptide consists of an aminoacid sequence set forth in SEQ ID NO:197. In some embodiments, thepeptide is a variant peptide designated M201. In one embodiment, thepeptide comprises an amino acid sequence set forth in SEQ ID NO:198. Inother embodiments, the peptide consists of an amino acid sequence setforth in SEQ ID NO:198. In other embodiments, the peptide is a variantpeptide designated M202. In some embodiments, the peptide comprises anamino acid sequence set forth in SEQ ID NO:199. In other embodiments,the peptide consists of an amino acid sequence set forth in SEQ IDNO:199. In certain embodiments, the peptide is a variant peptidedesignated M203. In one embodiment, the peptide comprises an amino acidsequence set forth in SEQ ID NO:200. In other embodiments, the peptideconsists of an amino acid sequence set forth in SEQ ID NO:200. In someembodiments, the peptide is a variant peptide designated M204. In oneembodiment, the peptide comprises an amino acid sequence set forth inSEQ ID NO:201. In other embodiments, the peptide consists of an aminoacid sequence set forth in SEQ ID NO:201. In another embodiment, thepeptide is a variant peptide designated M205. In some embodiments, thepeptide comprises an amino acid sequence set forth in SEQ ID NO:202. Inother embodiments, the peptide consists of an amino acid sequence setforth in SEQ ID NO:202. In other embodiments, the peptide is a variantpeptide designated M206. In some embodiments, the peptide comprises anamino acid sequence set forth in SEQ ID NO:203. In other embodiments,the peptide consists of an amino acid sequence set forth in SEQ IDNO:203. In yet other embodiments, the peptide is a variant peptidedesignated M207. In some embodiments, the peptide comprises an aminoacid sequence set forth in SEQ ID NO:204. In other embodiments, thepeptide consists of an amino acid sequence set forth in SEQ ID NO:204.

In various additional particular aspects, the N-terminus of the peptidesequence includes or consists of any of:

HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO: 160);DSSPLLQFGGQVRLRHLYTSG (M6-R) (amino acids 2-22 of SEQ ID NO: 6);RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID NO: 7);HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID NO: 8);HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ ID NO: 9);HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of SEQ ID NO: 10);RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27 of SEQ ID NO: 11);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R) (amino acids 2-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15) (amino acids 1-27 of SEQ ID NO: 15);RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO: 16);RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID NO: 17);RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ ID NO: 18);RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of SEQ ID NO: 19);RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27 of SEQ ID NO: 20);RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids 1-27 of SEQ ID NO: 21);RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino acids 1-27 of SEQ ID NO: 22);RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23) (amino acids 1-27 of SEQ ID NO: 23);RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24) (amino acids 1-27 of SEQ ID NO: 24);RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID NO: 25);RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ ID NO: 26);RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of SEQ ID NO: 27);RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26 of SEQ ID NO: 28);RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids 1-28 of SEQ ID NO: 29);RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino acids 1-29 of SEQ ID NO: 30);RHPIPDSSPLLQFGAQVRLRHLYTSG (M31) (amino acids 1-26 of SEQ ID NO: 31);RHPIPDSSPLLQFGDQVRLRHLYTSG (M32) (amino acids 1-26 of SEQ ID NO: 32);RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID NO: 33);RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ ID NO: 34);RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of SEQ ID NO: 35);RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26 of SEQ ID NO: 36);RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids 1-26 of SEQ ID NO: 37);RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino acids 1-26 of SEQ ID NO: 38);RHPIPDSSPLLQFGGQTRLRHLYTSG (M39) (amino acids 1-26 of SEQ ID NO: 39);RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40) (amino acids 1-28 of SEQ ID NO: 40);DAGPHVHYGWGDPIRLRHLYTSG (M74-R) (amino acids 2-24 of SEQ ID NO: 74);VHYGWGDPIRLRHLYTSG (M75-R) (amino acids 2-19 of SEQ ID NO: 75);RLRHLYTSG (M77-R) (amino acids 2-10 of SEQ ID NO: 77);RHPIPDSSPLLQFGWGDPIRLRHLYTSG (M9) (amino acids 1-28 of SEQ ID NO: 9);RHPIPDSSPLLQWGDPIRLRHLYTSG (M8) (amino acids 1-26 of SEQ ID NO: 8);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID NO: 10);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27 of SEQ ID NO: 43); orRDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22 of SEQ ID NO: 6);or any of the foregoing peptide sequences where the amino terminal Rresidue is deleted.

In certain embodiments, the peptide comprises or consists of any of:

HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO: 160);DSSPLLQFGGQVRLRHLYTSG (M6-R) (amino acids 2-22 of SEQ ID NO: 6);RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID NO: 7);HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID NO: 8);HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ ID NO: 9);HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of SEQ ID NO: 10);RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27 of SEQ ID NO: 11);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R) (amino acids 2-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15) (amino acids 1-27 of SEQ ID NO: 15);RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO: 16);RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID NO: 17);RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ ID NO: 18);RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of SEQ ID NO: 19);RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27 of SEQ ID NO: 20);RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids 1-27 of SEQ ID NO: 21);RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino acids 1-27 of SEQ ID NO: 22);RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23) (amino acids 1-27 of SEQ ID NO: 23);RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24) (amino acids 1-27 of SEQ ID NO: 24);RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID NO: 25);RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ ID NO: 26);RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of SEQ ID NO: 27);RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26 of SEQ ID NO: 28);RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids 1-28 of SEQ ID NO: 29);RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino acids 1-29 of SEQ ID NO: 30);RHPIPDSSPLLQFGAQVRLRHLYTSG (M31) (amino acids 1-26 of SEQ ID NO: 31);RHPIPDSSPLLQFGDQVRLRHLYTSG (M32) (amino acids 1-26 of SEQ ID NO: 32);RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID NO: 33);RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ ID NO: 34);RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of SEQ ID NO: 35);RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26 of SEQ ID NO: 36);RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids 1-26 of SEQ ID NO: 37);RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino acids 1-26 of SEQ ID NO: 38);RHPIPDSSPLLQFGGQTRLRHLYTSG (M39) (amino acids 1-26 of SEQ ID NO: 39);RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40) (amino acids 1-28 of SEQ ID NO: 40);DAGPHVHYGWGDPIRLRHLYTSG (M74-R) (amino acids 2-24 of SEQ ID NO: 74);VHYGWGDPIRLRHLYTSG (M75-R) (amino acids 2-19 of SEQ ID NO: 75);RLRHLYTSG (M77-R) (amino acids 2-10 of SEQ ID NO: 77);RHPIPDSSPLLQFGWGDPIRLRHLYTSG (M9) (amino acids 1-28 of SEQ ID NO: 9);RHPIPDSSPLLQWGDPIRLRHLYTSG (M8) (amino acids 1-26 of SEQ ID NO: 8);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID NO: 10);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27 of SEQ ID NO: 43); orRDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22 of SEQ ID NO: 6).In some embodiments, the peptide comprise one of the foregoingsequences. In another embodiment, the peptide consists of one of theforegoing sequences. In some embodiments, the peptide comprises aC-terminal region comprising a portion of SEQ ID NO:99 (FGF19), theC-terminal region having a first amino acid position and a last aminoacid position, wherein the C-terminal region comprises amino acidresidues 16-29 of SEQ ID NO:99 (FGF19), WGDPIRLRHLYTSG (SEQ ID NO:169),wherein the W residue corresponds to the first amino acid position ofthe C-terminal region.

In various further particular aspects, a peptide sequence includes orconsists of:

HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSF EK (SEQ ID NO: 160);DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 138 or 161);RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 1 or 139);RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 2 or 140); orDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 141);or a subsequence or fragment thereof any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the R terminal residue is deleted.

In further embodiments, a peptide sequence comprises or consists of:

MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70) (SEQ ID NO: 70),or a subsequence or fragment thereof.

In further embodiments, a peptide sequence comprises or consists of:

RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M69) (SEQ ID NO: 69),or a subsequence or fragment thereof.

In certain embodiments, a peptide sequence includes the addition ofamino acid residues 30-194 of SEQ ID NO:99 (FGF19) at the C-terminus,resulting in a chimeric polypeptide. In some embodiments, a peptidesequence has at least one amino acid substitution to amino acid residues125-129 of SEQ ID NO:99 (FGF19), EIRPD. In other embodiments, thepeptide sequence has at least one amino acid substitution to amino acidresidues 126-128 of SEQ ID NO:99 (FGF19), IRP. In other embodiments, thepeptide sequence has at least one amino acid substitution to amino acidresidues 127-128 of SEQ ID NO:99 (FGF19), RP. In other embodiments, thepeptide sequence has at least one amino acid substitution to amino acidresidues 1-124 of SEQ ID NO:99 (FGF19) and/or to amino acid residues130-194 of SEQ ID NO:99 (FGF19). For example, in certain embodiments, apeptide sequence comprises substitution to one of amino acid residues127-128 of SEQ ID NO:99 (FGF19), RP, wherein at least one amino acidsubstitution is R127L or P128E. Said substitutions within acorresponding FGF19 sequence (e.g., EIRPD, IRP or RP) of a peptidevariant provided herein is also contemplated. In certain embodiments,the peptide comprises both a R127L and P128E substitution to amino acidresidues 127-128 of SEQ ID NO:99 (FGF19), RP, or the corresponding FGF19sequence thereof in a variant peptide provided herein. In certainembodiments, the amino acid sequence of the peptide comprises at leastone amino acid substitution in the Loop-8 region of FGF19, or thecorresponding FGF19 sequence thereof in a variant peptide providedherein. In certain embodiments, the amino acid sequence of the peptidecomprises one amino acid substitution to the EIRPD (amino acids 2-6 ofSEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19. Insome embodiments, the amino acid sequence of the peptide comprises twoamino acid substitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190)amino acid sequence in the Loop-8 region of FGF19. In other embodiments,the amino acid sequence of the peptide comprises three amino acidsubstitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In certain embodiments, theamino acid sequence of the peptide comprises four amino acidsubstitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In some embodiments, the aminoacid sequence of the peptide comprises five amino acid substitutions tothe EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in theLoop-8 region of FGF19. In certain embodiments, the amino acid sequenceof the peptide comprises one amino acid substitution to the IRP (aminoacids 3-5 of SEQ ID NO:190) amino acid sequence in the Loop-8 region ofFGF19. In some embodiments, the amino acid sequence of the peptidecomprises two amino acid substitutions to the IRP (amino acids 3-5 ofSEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19. Inother embodiments, the amino acid sequence of the peptide comprisesthree amino acid substitutions to the IRP (amino acids 3-5 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19. In certainembodiments, the amino acid sequence of the peptide comprises one aminoacid substitution to the RP (amino acids 4-5 of SEQ ID NO:190) aminoacid sequence in the Loop-8 region of FGF19. In some embodiments, theamino acid sequence of the peptide comprises two amino acidsubstitutions to the RP (amino acids 4-5 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In certain embodiments, theamino acid substitution to the RP (amino acids 4-5 of SEQ ID NO:190)amino acid sequence in the Loop-8 region of FGF19 is an Arg (R) to Leu(L) substitution. In other embodiments, the substitution to the RP(amino acids 4-5 of SEQ ID NO:190) amino acid sequence in the Loop-8region of FGF19 is a Pro (P) to Glu (E) substitution. In someembodiments, the substitutions to the RP (amino acids 4-5 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19 is an Arg (R)to Leu (L) substitution and a Pro (P) to Glu (E) substitution. Inspecific embodiments, the foregoing substitution(s) in the Loop-8 regionof FGF19 is in the corresponding FGF19 sequence thereof in a variantpeptide provided herein. That is, said substitutions within acorresponding FGF19 sequence (e.g., EIRPD, IRP or RP) of a peptidevariant provided herein is also contemplated.

Peptide or chimeric sequences provided herein can be of any suitablelength. In particular embodiments, the N-terminal or C-terminal regionof the peptide or chimeric sequence is from about 20 to about 200 aminoacid residues in length. In further particular embodiments, a chimericpeptide sequence or peptide sequence has at least one amino aciddeletion. In other particular aspects, a peptide or chimeric sequencehas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or more amino acid deletions from the amino terminus, thecarboxy-terminus or internally. In one embodiment, the amino acidsubstitution, or deletion is at any of amino acid positions 8-20 ofFGF19 (AGPHVHYGWGDPI) (SEQ ID NO:187). In further particularembodiments, a peptide or chimeric sequence has an N-terminal region, ora C-terminal region that comprises or consists of an amino acid sequenceof about 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70to 80, 80 to 90, 90 to 100 or more amino acids. In additional moreparticular embodiments, a peptide or chimeric sequence has a FGF19sequence portion, or a FGF21 sequence portion that comprises or consistsof an amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or moreamino acids of FGF19 or FGF21.

In various further embodiments, a peptide or chimeric sequence has anamino acid substitution, an addition, insertion or is a subsequence thathas at least one amino acid deleted. Such amino acid substitutions,additions, insertions and deletions of a peptide sequence can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues (10-20, 20-30,30-40, 40-50, etc.), for example, at the N- or C-terminus, or internal.For example, a subsequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid deletions from theamino terminus, the carboxy-terminus or internally. In a particularaspect, the amino acid substitution, or deletion is at any of amino acidpositions 8-20 of FGF19 (AGPHVHYGWGDPI) (SEQ ID NO:187).

In various still more particular embodiments, a peptide or chimericsequence includes all or a portion of a FGF19 sequence set forth as:

PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188)positioned at the C-terminus of the peptide, or the amino terminal “R”residue is deleted from the sequence.

In various embodiments, a peptide or chimeric sequence has a function oractivity greater or less than a comparison sequence. In furtherparticular embodiments, chimeric peptide sequences and peptide sequenceshave particular functions or activities. In one aspect, a chimericpeptide sequence or peptide sequence maintains or increases a fibroblastgrowth factor receptor 4 (FGFR4) mediated activity. In additionalaspects, a chimeric peptide sequence or peptide sequence binds to FGFR4or activates FGFR4, or does not detectably bind to FGFR4 or activateFGFR4, or binds to FGFR4 with an affinity less than, comparable to orgreater than FGF19 binding affinity for FGFR4, or activates FGFR4 to anextent or amount less than, comparable to or greater than FGF19activates FGFR4. In some embodiments, a chimeric peptide sequence orpeptide sequence provided herein activates FGFR4 to an extent or amountless than the extent or amount that FGF19 activates FGFR4. In oneembodiment, a chimeric peptide sequence or peptide sequence providedherein activates FGFR4 to an extent or amount comparable to the extentor amount that FGF19 activates FGFR4. In one embodiment, a chimericpeptide sequence or peptide sequence provided herein activates FGFR4 toan extent or amount greater than the extent or amount that FGF19activates FGFR4.

In one embodiment, a chimeric peptide sequence or peptide sequenceprovided herein maintains a FGFR4 mediated activity. In one embodiment,a chimeric peptide sequence or peptide sequence provided hereinincreases a FGFR4 mediated activity. In some embodiments, a chimericpeptide sequence or peptide sequence provided herein binds to FGFR4 withan affinity less than FGF19 binding affinity for FGFR4. In oneembodiment, a chimeric peptide sequence or peptide sequence providedherein binds to FGFR4 with an affinity comparable to FGF19 bindingaffinity for FGFR4. In some embodiments, a chimeric peptide sequence orpeptide sequence provided herein binds to FGFR4 with an affinity greaterthan FGF19 binding affinity for FGFR4. In one embodiment, a chimericpeptide sequence or peptide sequence provided herein does not detectablybind to FGFR4.

In further aspects, a chimeric peptide sequence or peptide sequence hasreduced HCC formation compared to FGF19, or a FGF19 variant sequencehaving any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172),WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175),WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178),WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI(SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184)substituted for the WGDPI (SEQ ID NO:170) sequence at amino acids 16-20of FGF19; or has greater glucose lowering activity compared to FGF19, ora FGF19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID NO:170) sequenceat amino acids 16-20 of FGF19; has less lipid increasing activitycompared to FGF19, or a FGF19 variant sequence having any of GQV, GDI,WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI(SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176),AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179),WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP(SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQID NO:170) sequence at amino acids 16-20 of FGF19; or has lesstriglyceride, cholesterol, non-HDL or HDL increasing activity comparedto FGF19, or a FGF19 variant sequence having any of GQV, GDI, WGPI (SEQID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19; or the peptide sequencehas less lean mass reducing activity compared to FGF21. Such functionsand activities can be ascertained in vitro or in vivo, for example, in adb/db mouse.

In one embodiment, a peptide or chimeric sequence has a function oractivity greater or less than a comparison sequence. In someembodiments, the comparison sequence is FGF19. In another embodiment,the comparison sequence is FGF19 variant sequence having any of GQV,GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173),GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176),AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179),WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP(SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQID NO:170) sequence at amino acids 16-20 of FGF19. In one embodiment, apeptide or chimeric peptide sequence provided herein has greater glucoselowering activity compared to a comparison sequence. In anotherembodiment, a peptide or chimeric peptide sequence provided herein hasless lipid increasing activity compared to a comparison sequence. Inother embodiment, a peptide or chimeric peptide sequence provided hereinhas lower or reduced lipid (e.g., triglyceride, cholesterol, non-HDL)activity compared to a comparison sequence. In other embodiments, apeptide or chimeric peptide sequence provided herein has more HDLincreasing activity as compared to a comparison sequence. In otherembodiment, a peptide or chimeric peptide sequence provided herein hasless lean mass reducing activity compared to a comparison sequence orFGF21.

In further additional various embodiments, a peptide or chimericsequence includes one or more L-amino acids, D-amino acids,non-naturally occurring amino acids, or amino acid mimetic, derivativeor analogue. In still further various embodiments, a peptide or chimericsequence has an N-terminal region, or a C-terminal region, or a FGF19sequence portion, or a FGF21 sequence portion, joined by a linker orspacer.

In certain embodiments, a pharmaceutical composition provided herein isin liquid dosage form. In some embodiments, the peptide is at aconcentration of from 1 to 10 mg/mL. In one embodiment, the peptide isat a concentration of 1 mg/mL. In another embodiment, the peptide is ata concentration of 5 mg/mL. In some embodiments, the peptide at aconcentration of 10 mg/mL. In one embodiment, the peptide comprises anamino acid sequence of SEQ ID NO:70. In some embodiments, the peptideconsists of an amino acid sequence of SEQ ID NO:70. In one embodiment,the peptide comprises or consists of an amino acid sequence of SEQ IDNO:204.

In some embodiments of the pharmaceutical compositions provided herein,Tris is present in the range of 5 and 50 mM, 15 and 40 mM, 20 and 35 mM,20 and 30 mM, or 20 and 25 mM. In one embodiment, the Tris is present at20 mM. In some embodiments, a formulation volume of 1000 mL comprises2.0, 2.1, 2.2., 2.3., 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0 g of Tris, orany range thereof. In one embodiment, the a formulation volume of 1000mL comprises from 2.4 to 2.5 g of Tris.

In some embodiments of the various pharmaceutical compositions providedherein, trehalose is present in the range of 1 and 20%, 2 and 15%, 3 and10%, 4 and 9.5%, 5 and 9.25%, 6 and 9%, 7 and 8.5%, 8 and 8.4%, or 8.1and 8.3%. In one embodiment, the trehalose is present at 8.2%, 8.3% or8.4%. In some embodiments, a formulation volume of 1000 mL comprises92.0, 92.1, 92.2, 92.3, 92.4, 92.5, 92.6, 92.7, 92.8, 92.9 or 93.0 g oftrehalose dihydrate. In one embodiment, a formulation volume of 1000 mLcomprises from 92.4 to 92.5 g of trehalose dihydrate. In someembodiments, the molarity of trehalose dehydrate is from 240 to 300 mM,240 to 290 mM, 250 to 290 mM, 260 to 290 mM, 270 to 290 mM, or 244 to245 mM. In one embodiment, the molarity of the trehalose dehydrate isabout 280 mM.

In some embodiments of the various pharmaceutical compositions providedherein, the concentration of TWEEN-20 is in the range of from 0.001 to0.1% (v/v), 0.0025 to 0.075% (v/v), 0.005 to 0.05% (v/v), or 0.0075 to0.025% (v/v). In one embodiment, the concentration of TWEEN-20 is 0.01%(v/v).

In some embodiments of the various pharmaceutical compositions providedherein, the pharmaceutical composition has a pH in a range of 6.5 and9.5, 6.8 and 9.3, 7.0 and 9.0, 7.3 and 8.7, 7.5 and 8.5, or 7.7 and 8.2.In one embodiment, the pharmaceutical composition has a pH of 7.3, 7.5,8.0, or 8.5. In some embodiments, the pH is taken at 4° C. In oneembodiment, the pH is taken at 25° C.

In some embodiments of the various pharmaceutical compositions providedherein, the compositions comprise 20 mM Tris, 8.37% (w/v) trehalose, and0.01% TWEEN-20. In one embodiment of the various pharmaceuticalcompositions provided herein, the compositions comprise 20 mM Tris, 8.3%(w/v) trehalose, and 0.01% TWEEN-20. In one embodiment of the variouspharmaceutical compositions provided herein, the compositions comprise20 mM Tris, 8.2% (w/v) trehalose, and 0.01% TWEEN-20. In someembodiments of the various pharmaceutical compositions provided herein,the compositions comprise 20 mM Tris, 280 mM trehalose, and 0.01%TWEEN-20. In one embodiment, the pH is 8.0 at 25° C. In someembodiments, the polypeptide comprises SEQ ID NO:70. In otherembodiments, the polypeptide consists of SEQ ID NO:70. In someembodiments, the polypeptide comprises SEQ ID NO:69. In otherembodiments, the polypeptide consists of SEQ ID NO:69. In someembodiments, the polypeptide is fused to a human antibody Fc fragment.

Also provided herein is a pharmaceutical composition comprising: (A) apeptide comprising or consisting of SEQ ID NO:70, and (B) apharmaceutically acceptable carrier comprising: (i) 20 mM Tris, (ii)from 8.2% to 8.4% (w/v) trehalose, and (iii) 0.01% TWEEN-20. Alsoprovided herein is a pharmaceutical composition comprising: (A) apeptide comprising or consisting of SEQ ID NO:69, and (B) apharmaceutically acceptable carrier comprising: (i) 20 mM Tris, (ii)from 8.2% to 8.4% (w/v) trehalose, and (iii) 0.01% TWEEN-20. Alsoprovided herein is a pharmaceutical composition comprising: (A) apeptide comprising or consisting of SEQ ID NO:70, and (B) apharmaceutically acceptable carrier comprising: (i) 20 mM Tris, (ii)from 8.37% (w/v) trehalose, and (iii) 0.01% TWEEN-20. Also providedherein is a pharmaceutical composition comprising: (A) a peptidecomprising or consisting of SEQ ID NO:69, and (B) a pharmaceuticallyacceptable carrier comprising: (i) 20 mM Tris, (ii) from 8.37% (w/v)trehalose, and (iii) 0.01% TWEEN-20. Also provided herein is apharmaceutical composition comprising: (A) a peptide comprising orconsisting of SEQ ID NO:70, and (B) a pharmaceutically acceptablecarrier comprising: (i) 20 mM Tris, (ii) from 8.3% (w/v) trehalose, and(iii) 0.01% TWEEN-20. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:69, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.3% (w/v) trehalose, and (iii) 0.01% TWEEN-20. Alsoprovided herein is a pharmaceutical composition comprising: (A) apeptide comprising or consisting of SEQ ID NO:70, and (B) apharmaceutically acceptable carrier comprising: (i) 20 mM Tris, (ii)from 8.2% (w/v) trehalose, and (iii) 0.01% TWEEN-20. Also providedherein is a pharmaceutical composition comprising: (A) a peptidecomprising or consisting of SEQ ID NO:69, and (B) a pharmaceuticallyacceptable carrier comprising: (i) 20 mM Tris, (ii) from 8.2% (w/v)trehalose, and (iii) 0.01% TWEEN-20. In some embodiments, thepolypeptide comprises SEQ ID NO:70. In other embodiments, thepolypeptide consists of SEQ ID NO:70. In some embodiments, thepolypeptide comprises SEQ ID NO:69. In other embodiments, thepolypeptide consists of SEQ ID NO:69. In some embodiments, thepolypeptide is fused to a human antibody Fc fragment.

Also provided herein is a pharmaceutical composition comprising: (A) apeptide comprising or consisting of SEQ ID NO:70, and (B) apharmaceutically acceptable carrier comprising: (i) 20 mM Tris, (ii)from 8.2% to 8.4% (w/v) trehalose, and (iii) 0.01% TWEEN-20; wherein thepH is 8.0 at 25° C. Also provided herein is a pharmaceutical compositioncomprising: (A) a peptide comprising or consisting of SEQ ID NO:69, and(B) a pharmaceutically acceptable carrier comprising: (i) 20 mM Tris,(ii) from 8.2% to 8.4% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:70, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.37% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:69, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.37% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:70, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.3% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:69, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.3% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:70, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.2% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. Also provided herein is a pharmaceuticalcomposition comprising: (A) a peptide comprising or consisting of SEQ IDNO:69, and (B) a pharmaceutically acceptable carrier comprising: (i) 20mM Tris, (ii) from 8.2% (w/v) trehalose, and (iii) 0.01% TWEEN-20;wherein the pH is 8.0 at 25° C. In some embodiments, the polypeptidecomprises SEQ ID NO:70. In other embodiments, the polypeptide consistsof SEQ ID NO:70. In some embodiments, the polypeptide comprises SEQ IDNO:69. In other embodiments, the polypeptide consists of SEQ ID NO:69.In some embodiments, the polypeptide is fused to a human antibody Fcfragment.

In some embodiments of the various pharmaceutical compositions providedherein, the pharmaceutical composition does not comprise a salt. In oneembodiment, the pharmaceutical composition does not comprise NaCl.

In some embodiments of the various pharmaceutical compositions providedherein, the pharmaceutical composition is in a liquid form. In certainembodiments, the pharmaceutical composition is lyophilized.

In one embodiment of the various pharmaceutical compositions providedherein, the compositions comprise less than 20%, less than 15%, lessthan 10%, less than 5% or less than 2% of peptide aggregates after aperiod of time. In some embodiments, the aggregates are measured usingan assay provided herein (e.g., in the Experimental section). In oneembodiment, the composition comprises less than 20% of peptideaggregates after a period of time. In one embodiment, the compositioncomprises less than 15% of peptide aggregates after a period of time. Inone embodiment, the composition comprises less than 10% of peptideaggregates after a period of time. In one embodiment, the compositioncomprises less than 5% of peptide aggregates after a period of time. Inone embodiment, the composition comprises less than 2% of peptideaggregates after a period of time. In one embodiment of the variouspharmaceutical compositions provided herein, the compositions compriseless than 20%, less than 15%, less than 10%, less than 5% or less than2% of peptide precipitates after a period of time. In some embodiments,the precipitates are measured using an assay provided herein (e.g., inthe Experimental section). In one embodiment, the composition comprisesless than 20% of peptide precipitates after a period of time. In oneembodiment, the composition comprises less than 15% of peptideprecipitates after a period of time. In one embodiment, the compositioncomprises less than 10% of peptide precipitates after a period of time.In one embodiment, the composition comprises less than 5% of peptideprecipitates after a period of time. In one embodiment, the compositioncomprises less than 2% of peptide precipitates after a period of time.In certain embodiments. the period of time is 1 day, 7 days, 14 days, 28days, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11, months, 12 months, 18 months, 24 monthsor longer. In one embodiment, the period of time is 1 day. In oneembodiment, the period of time is 7 days. In one embodiment, the periodof time is 14 days. In one embodiment, the period of time is 21 days. Inone embodiment, the period of time is 1 month. In one embodiment, theperiod of time is 2 months. In one embodiment, the period of time is 3months. In one embodiment, the period of time is 4 months. In oneembodiment, the period of time is 5 months. In one embodiment, theperiod of time is 6 months. In one embodiment, the period of time is 7months. In one embodiment, the period of time is 8 months. In oneembodiment, the period of time is 9 months. In one embodiment, theperiod of time is 10 months. In one embodiment, the period of time is 11months. In one embodiment, the period of time is 12 months. In oneembodiment, the period of time is 18 months. In one embodiment, theperiod of time is 24 months. In some embodiments, the pharmaceuticalcomposition is stored at −80° C. In one embodiment, the pharmaceuticalcomposition is stored at 4° C. In some embodiments, the pharmaceuticalcomposition is stored at 25° C. In one embodiment, the pharmaceuticalcomposition is stored at 37° C.

In certain embodiments, a pharmaceutical composition provided herein hasa shelf life of at least about 12 months, at least about 24 months or atleast about 36 months. In certain embodiments, a pharmaceuticalcomposition provided herein has a shelf life of at least about 12months. In certain embodiments, a pharmaceutical composition providedherein has a shelf life of at least about 24 months. In certainembodiments, a pharmaceutical composition provided herein has a shelflife of at least about 36 months. In some embodiments, thepharmaceutical composition is stored at −80° C. In one embodiment, thepharmaceutical composition is stored at 4° C. In some embodiments, thepharmaceutical composition is stored at 25° C. In one embodiment, thepharmaceutical composition is stored at 37° C.

In still additional embodiments, the chimeric peptide or peptidesequence is included in a pharmaceutical composition, which in turn canbe used for practicing the methods and uses provided herein. Suchcompositions include combinations of inactive or other activeingredients. In one embodiment, a composition, such as a pharmaceuticalcomposition includes chimeric peptide sequence or peptide sequence andan agent, for example, that improves bile acid homeostasis

In one embodiment, provided herein is a method of preventing a diseaseor disorder in a subject having, or at risk of having, a disease ordisorder preventable by a peptide sequence provided herein, comprisingadministering a pharmaceutical composition comprising a peptide providedherein to a subject in an amount effective for preventing the disease ordisorder. In another embodiment, provided herein is a method of treatinga disease or disorder in a subject having, or at risk of having, adisease or disorder treatable by a peptide sequence provided herein,comprising administering a pharmaceutical composition comprising apeptide provided herein to a subject in an amount effective for treatingthe disease or disorder. In yet another embodiment, provided herein is amethod of managing a disease or disorder in a subject having, or at riskof having, a disease or disorder manageable by a peptide sequenceprovided herein, comprising administering a pharmaceutical compositioncomprising a peptide provided herein to a subject in an amount effectivefor managing the disease or disorder. In one embodiment, the disease ordisorder is a bile acid-related disease or associated disorder. Inanother embodiment, the disease or disorder is a metabolic disease ordisorder. In other embodiments, the disease or disorder is a cancer ortumor.

Non-limiting exemplary bile acid-related or associated disorderspreventable, treatable or manageable according to the methods and usesprovided herein include: cholestasis, including, for example diseases ofintrahepatic cholestasis (e.g., primary biliary cirrhosis (PBC), primaryfamilial intrahepatic cholestasis (PFIC) (e.g., progressive PFIC),primary sclerosing choangitis (PSC), pregnancy intrahepatic cholestasis(PIC), neonatal cholestasis, and drug-induced cholestasis (e.g.,estrogen)), and diseases of extrahepatic cholestasis (e.g., bile cutcompression from tumor, bile duct blockade by gall stones); bile acidmalabsorption and other disorders involving the distal small intestine,including ileal resection, inflammatory bowel diseases (e.g., Crohn'sdisease and ulcerative colitis), short bowel syndrome, disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., bile acid diarrhea (BAD)) andGI symptoms, and GI, liver, and/or biliary cancers (e.g., colon cancerand hepatocellular cancer); and/or bile acid synthesis abnormalities,such as those contributing to non-alcoholic steatohepatitis (NASH),cirrhosis and portal hypertension; e.g., in mammals, such as humans.Additional bile acid-related or associated disorders include metabolicsyndrome; a lipid or glucose disorder; cholesterol or triglyceridemetabolism; type 2 diabetes. In one particular embodiment, the bileacid-related or associated disorder is bile acid malabsorption. Inanother particular embodiment, the bile acid-related or associateddisorder is diarrhea. In a still further particular embodiment, the bileacid-related or associated disorder is cholestasis (e.g., intrahepaticor extrahepatic cholestasis). In another further particular embodiment,the bile acid-related or associated disorder is primary biliarycirrhosis (PBC). In other particular embodiments, the bile acid-relatedor associated disorder is primary sclerosing cholangitis. In anotherembodiment, the bile acid-related or associated disorder is PFIC (e.g.,progressive PFIC). In another embodiment, the bile acid-related orassociated disorder is NASH. In another embodiment, the bileacid-related or associated disorder is a hyperglycemic condition. In aspecific embodiment, the bile acid-related or associated disorder istype 2 diabetes.

In some embodiments, the pharmaceutical composition further comprises atleast one additional agent effective in modulating bile acid homeostasisor treating a bile acid-related or associated disorder, wherein theadditional agent is: a glucocorticoid; CDCA; UDCA; insulin, an insulinsecretagogues, an insulin mimetic, a sulfonylurea and a meglitinide; abiguanide; an alpha-glucosidase inhibitors; a DPP-IV inhibitor, GLP-1, aGLP-1 agonists and a GLP-1 analog; a DPP-IV-resistant analogue; a PPARgamma agonist, a dual-acting PPAR agonist, a pan-acting PPAR agonist; aPTP1B inhibitor; an SGLT inhibitor; an RXR agonist; a glycogen synthasekinase-3 inhibitor; an immune modulator; a beta-3 adrenergic receptoragonist; an 11beta-HSD1 inhibitor; amylin and an amylin analogue; a bileacid sequestrant; or an SGLT-2 inhibitor. In certain embodiments, the atleast one additional agent effective in modulating PBC is UDCA, an FXRagonist, OCA, an ASBT inhibitor, an autoimmune agent, an anti-IL-12agent, an anti-CD80 agent, an anti-CD20 agent, a CXCL10 neutralizingantibody, a ligand for CXCR3, a fibrate, fish oil, colchicine,methotrexate, azathioprine, cyclosporine, or an anti-retroviral therapy.In particular embodiments, the at least one additional agent effectivein modulating PBC is UDCA, OCA, an ASBT inhibitor, an anti-IL-12 agent,an anti-CD20 agent, or a fibrate.

Non-limiting exemplary disorders or conditions preventable, treatable ormanageable with the peptide formulations, methods and uses thereofprovided herein, include metabolic diseases and disorders. Non limitingexamples of diseases and disorders include: metabolic syndrome; a lipid-or glucose-related disorder; cholesterol or triglyceride metabolism;type 2 diabetes; cholestasis, including, for example diseases ofintrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatalcholestasis, and drug induced cholestasis (e.g., estrogen)), anddiseases of extrahepatic cholestasis (e.g., bile cut compression fromtumor, bile duct blockade by gall stones); bile acid malabsorption andother disorders involving the distal small intestine, including ilealresection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension. For treatment, peptide provided herein can be administeredto subjects in need of modulation of bile acid homeostasis or having abile-acid related or associated disorder. Peptides provided herein mayalso be useful in other hyperglycemic-related disorders, includingkidney damage (e.g., tubule damage or nephropathy), liver degeneration,eye damage (e.g., diabetic retinopathy or cataracts), and diabetic footdisorders; dyslipidemias and their sequelae such as, for example,atherosclerosis, coronary artery disease, cerebrovascular disorders andthe like.

Other conditions which may be associated with metabolic syndrome, suchas obesity and elevated body mass (including the co-morbid conditionsthereof such as, but not limited to, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovariansyndrome (PCOS), and also include thromboses, hypercoagulable andprothrombotic states (arterial and venous), hypertension (includingportal hypertension (defined as a hepatic venous pressure gradient(HVPG) greater than 5 mm Hg), cardiovascular disease, stroke and heartfailure; Disorders or conditions in which inflammatory reactions areinvolved, including atherosclerosis, chronic inflammatory bowel diseases(e.g., Crohn's disease and ulcerative colitis), asthma, lupuserythematosus, arthritis, or other inflammatory rheumatic disorders;Disorders of cell cycle or cell differentiation processes such asadipose cell tumors, lipomatous carcinomas including, for example,liposarcomas, solid tumors, and neoplasms; Neurodegenerative diseasesand/or demyelinating disorders of the central and peripheral nervoussystems and/or neurological diseases involving neuroinflammatoryprocesses and/or other peripheral neuropathies, including Alzheimer'sdisease, multiple sclerosis, Parkinson's disease, progressive multifocalleukoencephalopathy and Guillian-Barre syndrome; Skin and dermatologicaldisorders and/or disorders of wound healing processes, includingerythemato-squamous dermatoses; and other disorders such as syndrome X,osteoarthritis, and acute respiratory distress syndrome.

In some embodiments, the disease or disorder is a cancer or tumor. Incertain embodiments, the cancer or tumor is a FGF19-dependent cancer ortumor. In one embodiment, the FGF19-dependent cancer or tumor ishepatocellular carcinoma. In some embodiments, the FGF19 cancer or tumoris not hepatocellular carcinoma. In one embodiment, the cancer or tumoris a colon cancer or tumor. In some embodiments, the cancer or tumor isa prostate cancer or tumor. In one embodiment, the cancer or tumor is alung cancer or tumor. In some embodiments, the method results in areduction in tumor number, tumor size, or tumor weight.

In one embodiment, of the various methods provided herein, the subjectis a human. In certain embodiments, the subject is a subject in needthereof.

In some embodiments, the chimeric peptide sequence or a peptide sequencedescribed herein, either alone or in combination with at least oneadditional therapeutic agent or treatment modality, is assessed toensure that it does not cause untoward adverse effects in the subject.In a particular aspect, the combination of a chimeric peptide sequenceor a peptide sequence described herein and at least one additionaltherapeutic agent or treatment modality is assessed to ensure that itdoes not induce HCC in the subject. Such assessments may be performedbefore initiation of therapy (e.g., in a dose escalation study), duringtherapy, (e.g., by evaluating a marker correlating with HCC activity),or subsequent to termination of therapy (e.g., by performing a liverbiopsy). In some aspects, the assessment is performed in a suitable testenvironment (e.g., a validated animal model). One of ordinary skill inthe art is familiar with additional means for ensuring that thecombination therapy described herein is suitable for the particularsubject, or a subject population representative of the particularsubject, taking into consideration all relevant factors including, forexample, the severity of the subject's bile acid-related or associateddisorder (e.g., PBC) and the other medications be taken by the subject.

4. DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of visual observation of ten differentformulation buffers under the following conditions: concentration ofactive ingredient 1 mg/mL; temperature 37° C.; after 4 weeks. Sampleswere re-suspended prior to visual scoring and photo. Scoring system:0—Clear; 1—Slightly Hazy; 2—Slightly Cloudy; 3—Cloudy; 4—Very Cloudy;5—Extremely cloudy.

FIG. 2 depicts the results of visual observation of ten differentformulation buffers under the following conditions: concentration ofactive ingredient 10 mg/mL; temperature 37° C.; after 4 weeks. Sampleswere re-suspended prior to visual scoring and photo. Scoring system:0—Clear; 1—Slightly Hazy; 2—Slightly Cloudy; 3—Cloudy; 4—Very Cloudy;5—Extremely cloudy.

FIG. 3 depicts the results of HPLC analysis based on SEC AUC ofabsorbance at A₂₁₀ for 1 mg/mL samples, showing the percentage (%) ofmonomer at each time point.

FIG. 4 depicts the results of HPLC analysis based on SEC AUC ofabsorbance at A₂₁₀ in Buffer 7 for 1 mg/mL samples.

FIG. 5 depicts the results of HPLC analysis based on SEC AUC ofabsorbance at A₂₁₀ for 10 mg/mL samples, showing the percentage (%) ofmonomer at each time point.

FIG. 6 depicts the results of HPLC analysis based on SEC AUC ofabsorbance at A₂₁₀ in formulation Buffer 7 for 10 mg/mL samples.

FIGS. 7A-7B depict SDS-PAGE gel electrophoresis for formulation (A)Buffer 7 at 4 weeks for concentration of (A) 1 mg/mL, and (B) 10 mg/mL.

FIG. 8 depicts IEC-HPLC for Buffer 7 at 37° C., a concentration of 1mg/mL at t=0 weeks, 1 week, 2 weeks, and 4 weeks.

FIG. 9 depicts IEC-HPLC for Buffer 7 at 37° C., a concentration of 10mg/mL at t=0 weeks, 1 week, 2 weeks, and 4 weeks.

FIG. 10 depicts RP-HPLC for Buffer 7 at 37° C., a concentration of 1mg/mL at t=0 weeks, 1 week, 2 weeks, and 4 weeks.

FIG. 11 depicts RP-HPLC for Buffer 7 at 37° C., a concentration of 10mg/mL at t=0 weeks, 1 week, 2 weeks, and 4 weeks.

FIG. 12 depicts stability data after freeze/thaw cycles of an M70formulation comprising 20 mM Tris pH 8.0 (25° C.), 8.3% (w/v) trehalose,0.01% polysorbate-20 (TWEEN-20) (“TTP buffer”).

5. DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

5.1 Definitions

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering a polypeptide or a pharmaceuticalcomposition comprising a polypeptide) initiated after a disease,disorder or condition, or a symptom thereof, has been diagnosed,observed, and the like so as to eliminate, reduce, suppress, mitigate,or ameliorate, either temporarily or permanently, at least one of theunderlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (i.e., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other medical professional that a subject requires orwill benefit from treatment.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering a polypeptide or apharmaceutical composition comprising a polypeptide) initiated in amanner (e.g., prior to the onset of a disease, disorder, condition orsymptom thereof) so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof, generallyin the context of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other medical professional that a subjectrequires or will benefit from preventative care.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as a part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a disease,disorder or condition when administered to a patient. Thetherapeutically effective amount can be ascertained by measuringrelevant physiological effects. For example, in the case of ahyperglycemic condition, a lowering or reduction of blood glucose or animprovement in glucose tolerance test can be used to determine whetherthe amount of an agent is effective to treat the hyperglycemiccondition. For example, a therapeutically effective amount is an amountsufficient to reduce or decrease any level (e.g., a baseline level) offasting plasma glucose (FPG), wherein, for example, the amount issufficient to reduce a FPG level greater than 200 mg/dl to less than 200mg/dl, wherein the amount is sufficient to reduce a FPG level between175 mg/dl and 200 mg/dl to less than the starting level, wherein theamount is sufficient to reduce a FPG level between 150 mg/dl and 175mg/dl to less than the starting level, wherein the amount is sufficientto reduce a FPG level between 125 mg/dl and 150 mg/dl to less than thestarting level, and so on (e.g., reducing FPG levels to less than 125mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110mg/dl, etc.). Moreover, in the case of HbAIc levels, the effectiveamount is an amount sufficient to reduce or decrease levels by more thanabout 10% to 9%, by more than about 9% to 8%, by more than about 8% to7%, by more than about 7% to 6%, by more than about 6% to 5%, and so on.More particularly, a reduction or decrease of HbAIc levels by about0.1%, 0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%,5%, 10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is contemplated bythe present disclosure. The therapeutically effective amount can beadjusted in connection with the dosing regimen and diagnostic analysisof the subject's condition and the like.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., level ofglucose or insulin) or subjective parameter (e.g., a subject's feelingof well-being).

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the subject's ability to reduce, within about 120 minutes,the level of plasma glucose back to a level determined before the intakeof glucose.

Broadly speaking, the terms “diabetes” and “diabetic” refer to aprogressive disease of carbohydrate metabolism involving inadequateproduction or utilization of insulin, frequently characterized byhyperglycemia and glycosuria. The terms “pre-diabetes” and“pre-diabetic” refer to a state wherein a subject does not have thecharacteristics, symptoms and the like typically observed in diabetes,but does have characteristics, symptoms and the like that, if leftuntreated, can progress to diabetes. The presence of these conditionscan be determined using, for example, either the fasting plasma glucose(FPG) test or the oral glucose tolerance test (OGTT). Both usuallyrequire a subject to fast for at least 8 hours prior to initiating thetest. In the FPG test, a subject's blood glucose is measured after theconclusion of the fasting; generally, the subject fasts overnight andthe blood glucose is measured in the morning before the subject eats. Ahealthy subject would generally have a FPG concentration between about90 and about 100 mg/dl, a subject with “pre-diabetes” would generallyhave a FPG concentration between about 100 and about 125 mg/dl, and asubject with “diabetes” would generally have a FPG level above about 126mg/dl. In the OGTT, a subject's blood glucose is measured after fastingand again two hours after drinking a glucose-rich beverage. Two hoursafter consumption of the glucose-rich beverage, a healthy subjectgenerally has a blood glucose concentration below about 140 mg/dl, apre-diabetic subject generally has a blood glucose concentration about140 to about 199 mg/dl, and a diabetic subject generally has a bloodglucose concentration about 200 mg/dl or above. While the aforementionedglycemic values pertain to human subjects, normoglycemia, moderatehyperglycemia and overt hyperglycemia are scaled differently in murinesubjects. A healthy murine subject after a four-hour fast wouldgenerally have a FPG concentration between about 100 and about 150mg/dl, a murine subject with “pre-diabetes” would generally have a FPGconcentration between about 175 and about 250 mg/dl and a murine subjectwith “diabetes” would generally have a FPG concentration above about 250mg/dl.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously administered, isable to overcome the insulin resistance, in whole or in part, andproduce a biologic response.

The term “metabolic syndrome” refers to an associated cluster of traitsthat includes, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and dyslipidemiacharacterized by high triglycerides, low high density lipoprotein(HDL)-cholesterol, and high small dense low density lipoprotein (LDL)particles. Subjects having metabolic syndrome are at risk fordevelopment of type 2 diabetes and/or other disorders (e.g.,atherosclerosis).

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that is associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin can be manifested in thefollowing diseases, disorders and conditions: hyperglycemia, type IIdiabetes, gestational diabetes, type I diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, other metabolic disorders (such as metabolicsyndrome, which is also referred to as syndrome X), and obesity, amongothers. The polypeptides of the present disclosure, and compositionsthereof, can be used, for example, to achieve and/or maintain glucosehomeostasis, e.g., to reduce glucose level in the bloodstream and/or toreduce insulin level to a range found in a healthy subject.

The term “hyperglycemia”, as used herein, refers to a condition in whichan elevated amount of glucose circulates in the blood plasma of asubject relative to a healthy individual. Hyperglycemia can be diagnosedusing methods known in the art, including measurement of fasting bloodglucose levels as described herein.

The term “hyperinsulinemia”, as used herein, refers to a condition inwhich there are elevated levels of circulating insulin when,concomitantly, blood glucose levels are either elevated or normal.Hyperinsulinemia can be caused by insulin resistance which is associatedwith dyslipidemia, such as high triglycerides, high cholesterol, highlow-density lipoprotein (LDL) and low high-density lipoprotein (HDL);high uric acids levels; polycystic ovary syndrome; type II diabetes andobesity. Hyperinsulinemia can be diagnosed as having a plasma insulinlevel higher than about 2 μU/mL.

As used herein, the phrase “body weight disorder” and similar termsrefer to conditions associated with excessive body weight and/orenhanced appetite. Various parameters are used to determine whether asubject is overweight compared to a reference healthy individual,including the subject's age, height, sex and health status. For example,a subject can be considered overweight or obese by assessment of thesubject's Body Mass Index (BMI), which is calculated by dividing asubject's weight in kilograms by the subject's height in meters squared.An adult having a BMI in the range of ˜18.5 to ˜24.9 kg/m² is consideredto have a normal weight; an adult having a BMI between ˜25 and ˜29.9kg/m² can be considered overweight (pre-obese); and an adult having aBMI of ˜30 kg/m² or higher can be considered obese. Enhanced appetitefrequently contributes to excessive body weight. There are severalconditions associated with enhanced appetite, including, for example,night eating syndrome, which is characterized by morning anorexia andevening polyphagia often associated with insomnia, but which can berelated to injury to the hypothalamus.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusion proteins with heterologous andhomologous leader sequences, with or without N-terminus methionineresidues; immunologically tagged proteins; and the like. It will beappreciated that throughout this disclosure reference is made to aminoacids according to the single letter or three letter codes.

As used herein, the term “variant” encompasses naturally-occurringvariants (e.g., homologs and allelic variants) andnon-naturally-occurring variants (e.g., muteins). Naturally-occurringvariants include homologs, i.e., nucleic acids and polypeptides thatdiffer in nucleotide or amino acid sequence, respectively, from onespecies to another. Naturally-occurring variants include allelicvariants, i.e., nucleic acids and polypeptides that differ in nucleotideor amino acid sequence, respectively, from one individual to anotherwithin a species. Non-naturally-occurring variants include nucleic acidsand polypeptides that comprise a change in nucleotide or amino acidsequence, respectively, where the change in sequence is artificiallyintroduced, e.g., the change is generated in the laboratory or otherfacility by human intervention (“hand of man”).

The term “native”, in reference to FGF19, refers to biologically active,naturally-occurring FGF19, including biologically active,naturally-occurring FGF19 variants. The term includes the 194 amino acidhuman FGF19 mature sequence.

The terms “label”, “labeling” and the like, when use in the context of apolypeptide or nucleic acid (or antibody, as appropriate) of the presentdisclosure are meant to refer broadly to any means useful in, forexample, polypeptide purification, identification, isolation andsynthesis. Labels are generally covalently bound to the polypeptide ofinterest and can be introduced in any manner known in the art, includingattachment to a mature polypeptide (generally at the N- or C-terminus),incorporation during solid-phase peptide synthesis, or throughrecombinant means. Examples include, but are not limited to,fluorescence, biotinylation, and radioactive isotopes. Polypeptide andnucleic acid molecules can be labeled by both in vitro and in vivomethods. Labeling reagents and kits can be obtained from a number ofcommercial sources (e.g., Thermo Fischer Scientific, Rockford, Ill.; andMolecular Probes/Life Technologies; Grand Island, N.Y.).

The term “muteins” as used herein refers broadly to mutated recombinantproteins, i.e., a polypeptide comprising an artificially introducedchange in amino acid sequence, e.g., a change in amino acid sequencegenerated in the laboratory or other facility by human intervention(“hand of man”). These proteins usually carry single or multiple aminoacid substitutions and are frequently derived from cloned genes thathave been subjected to site-directed or random mutagenesis, or fromcompletely synthetic genes.

As used herein in reference to native human FGF19 or a FGF19 mutein, theterms “modified”, “modification” and the like refer to one or morechanges that enhance a desired property of human FGF19, anaturally-occurring FGF19 variant, or a FGF19 mutein, wherein thechange(s) does not alter the primary amino acid sequence of the FGF19.Such desired properties include, for example, enhancing solubility,prolonging the circulation half-life, increasing the stability, reducingthe clearance, altering the immunogenicity or allergenicity, improvingaspects of manufacturability (e.g., cost and efficiency), and enablingthe raising of particular antibodies (e.g., by introduction of uniqueepitopes) for use in detection assays. Changes to human FGF19, anaturally-occurring FGF19 variant, or a FGF19 mutein that can be carriedout include, but are not limited to, pegylation (covalent attachment ofone or more molecules of polyethylene glycol (PEG), or derivativesthereof); glycosylation (e.g., N-glycosylation), polysialylation andhesylation; albumin fusion; albumin binding through, for example, aconjugated fatty acid chain (acylation); Fc-fusion; and fusion with aPEG mimetic. Some particular embodiments entail modifications involvingpolyethylene glycol, other particular embodiments entail modificationsinvolving albumin, and still other particular modifications entailmodifications involving glycosylation.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

The term “probe” refers to a fragment of DNA or RNA corresponding to agene or sequence of interest, wherein the fragment has been labeledradioactively (e.g., by incorporating ³²P or ³⁵S) or with some otherdetectable molecule, such as biotin, digoxygen or fluorescein. Asstretches of DNA or RNA with complementary sequences will hybridize, aprobe can be used, for example, to label viral plaques, bacterialcolonies or bands on a gel that contain the gene of interest. A probecan be cloned DNA or it can be a synthetic DNA strand; the latter can beused to obtain a cDNA or genomic clone from an isolated protein by, forexample, microsequencing a portion of the protein, deducing the nucleicacid sequence encoding the protein, synthesizing an oligonucleotidecarrying that sequence, radiolabeling the sequence and using it as aprobe to screen a cDNA library or a genomic library.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, in the contextof a polypeptide, a “heterologous” polypeptide can include operablylinked amino acid sequences that are derived from differentpolypeptides. Similarly, in the context of a polynucleotide encoding achimeric polypeptide, a “heterologous” polynucleotide can includeoperably linked nucleic acid sequences that can be derived fromdifferent genes. Exemplary “heterologous” nucleic acids includeexpression constructs in which a nucleic acid comprising a codingsequence is operably linked to a regulatory element (e.g., a promoter)that is from a genetic origin different from that of the coding sequence(e.g., to provide for expression in a host cell of interest, which canbe of different genetic origin than the promoter, the coding sequence orboth). In the context of recombinant cells, “heterologous” can refer tothe presence of a nucleic acid (or gene product, such as a polypeptide)that is of a different genetic origin than the host cell in which it ispresent.

The term “operably linked” refers to linkage between molecules toprovide a desired function. For example, “operably linked” in thecontext of nucleic acids refers to a functional linkage between nucleicacid sequences. By way of example, a nucleic acid expression controlsequence (such as a promoter, signal sequence, or array of transcriptionfactor binding sites) can be operably linked to a second polynucleotide,wherein the expression control sequence affects transcription and/ortranslation of the second polynucleotide. In the context of apolypeptide, “operably linked” refers to a functional linkage betweenamino acid sequences (e.g., different domains) to provide for adescribed activity of the polypeptide.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” or “immediately C-terminal” refers to aposition of a first amino acid residue relative to a second amino acidresidue where the first and second amino acid residues are covalentlybound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” aFGF19 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring FGF19 polypeptide or aFGF19-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologues or variants ofreference amino acid or DNA sequences.

In the context of a polypeptide, the term “isolated” refers to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it can naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring, “isolated”indicates the polypeptide has been separated from an environment inwhich it was made by either synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., by ascientist or a clinician) so that a polypeptide of interest is presentin a) a greater concentration (e.g., at least 3-fold greater, at least4-fold greater, at least 8-fold greater, at least 64-fold greater, ormore) than the concentration of the polypeptide in the starting sample,such as a biological sample (e.g., a sample in which the polypeptidenaturally occurs or in which it is present after administration), or b)a concentration greater than the environment in which the polypeptidewas made (e.g., as in a bacterial cell).

“Substantially pure” indicates that a component (e.g., a polypeptide)makes up greater than about 50% of the total content of the composition,and typically greater than about 60% of the total polypeptide content.More typically, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the component of interest. In some cases, the polypeptide will makeup greater than about 90%, or greater than about 95% of the totalcontent of the composition.

The terms “measuring” or “assaying” and grammatical variations thereofare used interchangeably herein and refer to either qualitative orquantitative determinations, or both qualitative and quantitativedeterminations. When the terms are used in reference to detection, anymeans of assessing the relative amount is contemplated, including thevarious methods set forth herein and known in the art. For example, geneexpression can be assayed or measured by a Northern blot, Western blot,immunoprecipitation assay, or by measuring activity, function or amountof the expressed protein.

The terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer toglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, that is, theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that can be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations, which can include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

In the context of an antibody, the term “isolated” refers to an antibodythat has been separated and/or recovered from contaminant components ofits natural environment; such contaminant components include materialswhich might interfere with diagnostic or therapeutic uses for theantibody, and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes.

As used herein, the term “FGF19-dependent” and similar terms, as used inthe context of a disease, disorder or condition, refers to a disease,disorder or other condition that is caused all, or in part, by theexpression of FGF19. In certain embodiments, the expression of FGF19 isamplified as compared to a control. In some embodiments, the expressionof FGF19 is amplified 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or any numericalrange thereof. In some embodiments, the amplified expression of FGF19directly results in the disease, disorder or condition, or a symptomthereof. In other embodiments, the amplified expression of FGF19indirectly results in the disease disorder or condition, or a symptomthereof.

5.2 Pharmaceutical Compositions

In one aspect, provided herein are “pharmaceutical compositions,” whichinclude a peptide sequence (or sequences) provided herein, includingsubsequences, variants and modified forms of the exemplified peptidesequences (e.g., sequences listed in the Sequence Listing or Table 1),and one or more pharmaceutically acceptable or physiologicallyacceptable diluents, carriers or excipients. In certain embodiments, thepeptide sequences are provided in combination with, or separate from,one or more additional agents. Also provided is a composition comprisingsuch one or more additional agents and one or more pharmaceuticallyacceptable or physiologically acceptable diluents, carriers orexcipients. In particular embodiments, a peptide sequence or sequencesand an additional agent(s) are present in a therapeutically acceptableamount. The pharmaceutical compositions may be used in accordance withthe methods and uses provided herein. Thus, for example, thepharmaceutical compositions can be administered ex vivo or in vivo to asubject in order to practice treatment methods and uses provided herein.Pharmaceutical compositions provided herein can be formulated to becompatible with the intended method or route of administration;exemplary routes of administration are set forth herein.

In one embodiment, provided are pharmaceutical compositions and dosageforms of chimeric and peptide sequences that modulate bile acidhomeostasis and are able to treat a bile-acid related or associateddisorder. In another embodiment, provided are pharmaceuticalcompositions and dosage forms of chimeric and peptide sequences thatmodulate hyperglycemic condition, insulin resistance, hyperinsulinemia,glucose intolerance, metabolic syndrome, or related disorders. Inanother embodiment, provided are pharmaceutical compositions and dosageforms of chimeric and peptide sequences that modulate a cancer or tumor.

In some aspects, the pharmaceutical compositions may further compriseother therapeutically active agents or compounds disclosed herein orknown to the skilled artisan which can be used in the treatment orprevention of various diseases and disorders as set forth herein. As setforth above, the additional therapeutically active agents or compoundsmay be present in a separate pharmaceutical composition(s). Exemplarydosing parameters and regimens are described herein.

Pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the peptide sequences providedherein, including subsequences, variants and modified forms of theexemplified peptide sequences (e.g., sequences listed in the SequenceListing or Table 1) and one or more pharmaceutically and physiologicallyacceptable formulation agents. In certain embodiments, thepharmaceutical composition further comprises one or more additionalagents described herein.

In one embodiment, a pharmaceutical composition comprises a peptideprovided herein. In some embodiments, a pharmaceutical compositioncomprises a therapeutically effective amount of a peptide providedherein. In certain embodiments, the pharmaceutical composition comprisesa pharmaceutically acceptable or physiologically acceptable diluent,carrier or excipient. In a specific embodiment, the peptide comprises anamino acid sequence of SEQ ID NO:70. In another embodiment, the peptideconsists of an amino acid sequence of SEQ ID NO:70. In a specificembodiment, the peptide comprises an amino acid sequence of SEQ IDNO:204. In another embodiment, the peptide consists of an amino acidsequence of SEQ ID NO:204. In certain embodiments, the pharmaceuticalcomposition further comprises one or more additional agents describedherein.

In certain embodiments, the pharmaceutical composition comprises thepeptide at a concentration of from 0.1-100 mg/mL. In some embodiments,the pharmaceutical composition comprises the peptide at a concentrationof from 1 to 10 mg/mL. In certain embodiments, the pharmaceuticalcomposition comprises the peptide at a concentration of 1 mg/mL. Inanother embodiment, the peptide is at a concentration of 5 mg/mL. Incertain embodiments, the pharmaceutical composition comprises thepeptide at a concentration of 10 mg/mL.

Suitable pharmaceutically acceptable or physiologically acceptablediluents, carriers or excipients include, but are not limited to,antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives(e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl,p-hydroxybenzoate), emulsifying agents, suspending agents, dispersingagents, solvents, fillers, bulking agents, buffers, vehicles, diluents,and/or adjuvants. For example, a suitable vehicle may be physiologicalsaline solution or citrate buffered saline, possibly supplemented withother materials common in pharmaceutical compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. Those skilled in the art willreadily recognize a variety of buffers that could be used in thepharmaceutical compositions and dosage forms used herein. Typicalbuffers include, but are not limited to pharmaceutically acceptable weakacids, weak bases, or mixtures thereof. Buffer components also includewater soluble materials such as phosphoric acid, tartaric acids, lacticacid, succinic acid, citric acid, acetic acid, ascorbic acid, asparticacid, glutamic acid, and salts thereof.

In a specific embodiment, the pharmaceutical composition comprisespotassium phosphate. In one embodiment, the pharmaceutical compositioncomprises sodium chloride. In a another embodiment, the pharmaceuticalcomposition comprises Tromethamine (Tris). In other embodiments, thepharmaceutical composition comprises trehalose dihydrate. In anotherembodiment, the pharmaceutical composition comprises TWEEN-20(polysorbate 20). In a yet another embodiment, the pharmaceuticalcomposition comprises hydrochloric acid. In a certain embodiment, thepharmaceutical composition comprises water. Any combination of two,three, four, five, six or all seven of the foregoing components are alsocontemplated.

Thus, in one embodiment, the pharmaceutical composition comprisespotassium phosphate, sodium chloride, Tromethamine (Tris), Trehalosedehydrate, TWEEN-20, hydrochloric acid, water, or any combinationthereof. In a specific embodiment, the pharmaceutical compositioncomprises potassium phosphate, sodium chloride, Tromethamine (Tris),Trehalose dihydrate, TWEEN-20, hydrochloric acid, and water.

In one embodiment, the molarity of Tris useful in the pharmaceuticalcompositions provided herein is in the range of between 5 and 50 mM. Inanother embodiment, the molarity of Tris is in the range of 10 and 45mM. In another embodiment, the molarity of Tris is in the range of 15and 40 mM. In another embodiment, the molarity of Tris is in the rangeof 20 and 35 mM. In another embodiment, the molarity of Tris is in therange of 20 and 30 mM. In another embodiment, the molarity of Tris is inthe range of 20 and 25 mM. In one particular embodiment, the molarity ofTris is 20 mM. In certain embodiments, a formulation volume of 1000 mLcomprises from 2 to 3 g of Tris. In some embodiments, a formulationvolume of 1000 mL comprises 2.1, 2.2., 2.3., 2.4, 2.5, 2.6, 2.7, 2.8,2.9 or 3.0 g of Tris. In some embodiments, a formulation volume of 1000mL comprises 2.1 g of Tris. In some embodiments, a formulation volume of1000 mL comprises 2.2 g of Tris. In some embodiments, a formulationvolume of 1000 mL comprises 2.3 g of Tris. In some embodiments, aformulation volume of 1000 mL comprises 2.4 g of Tris. In someembodiments, a formulation volume of 1000 mL comprises 2.5 g of Tris. Insome embodiments, a formulation volume of 1000 mL comprises 2.6 g ofTris. In some embodiments, a formulation volume of 1000 mL comprises 2.7g of Tris. In some embodiments, a formulation volume of 1000 mLcomprises 2.8 g of Tris. In some embodiments, a formulation volume of1000 mL comprises 2.9 g of Tris. In some embodiments, a formulationvolume of 1000 mL comprises 3.0 g of Tris. In some embodiments, aformulation volume of 1000 mL comprises from 2.4 to 2.5 g of Tris.

In one embodiment, the concentration of trehalose dihydrate useful inthe pharmaceutical compositions provided herein is in the range ofbetween 1% and 20% (w/v). In another embodiment, the concentration oftrehalose dihydrate is in the range of 2% and 15% (w/v). In oneembodiment, the concentration of trehalose dihydrate is in the range of3% and 10% (w/v). In another embodiment, the concentration of trehalosedihydrate is in the range of 4% and 9.5% (w/v). In one embodiment, theconcentration of trehalose dihydrate is in the range of 5% and 9.25%(w/v). In another embodiment, the concentration of trehalose dihydrateis in the range of 6% and 9% (w/v). In one embodiment, the concentrationof trehalose dihydrate is in the range of 7% and 8.5% (w/v). In anotherembodiment, the concentration of trehalose dihydrate is in the range of8% and 8.4% (w/v). In one embodiment, the concentration of trehalosedihydrate is in the range of 8.1 and 8.3% (w/v). In one particularembodiment, the concentration of trehalose dihydrate is 8.2%. In oneparticular embodiment, the concentration of trehalose dihydrate is 8.3%(w/v). In certain embodiments, a formulation volume of 1000 mL comprisesfrom 92 to 93 g of trehalose dihydrate. In some embodiments, aformulation volume of 1000 mL comprises 92.0, 92.1, 92.2, 92.3, 92.4,92.5, 92.6, 92.7, 92.8, 92.9 or 93.0 g of trehalose dihydrate. In someembodiments, a formulation volume of 1000 mL comprises 92.0 g oftrehalose dihydrate. In some embodiments, a formulation volume of 1000mL comprises 92.1 g of trehalose dihydrate. In some embodiments, aformulation volume of 1000 mL comprises 92.2 g of trehalose dihydrate.In some embodiments, a formulation volume of 1000 mL comprises 92.3 g oftrehalose dihydrate. In some embodiments, a formulation volume of 1000mL comprises 92.4 g of trehalose dihydrate. In some embodiments, aformulation volume of 1000 mL comprises 92.5 g of trehalose dihydrate.In some embodiments, a formulation volume of 1000 mL comprises 92.6 g oftrehalose dihydrate. In some embodiments, a formulation volume of 1000mL comprises 92.7 g of trehalose dihydrate. In some embodiments, aformulation volume of 1000 mL comprises 92.8 g of trehalose dihydrate.In some embodiments, a formulation volume of 1000 mL comprises 92.9 g oftrehalose dihydrate. In some embodiments, a formulation volume of 1000mL comprises 93.0 g of trehalose dihydrate. In some embodiments, aformulation volume of 1000 mL comprises from 92.4 to 92.5 g of trehalosedihydrate. In certain embodiments, the molarity of the trehalosedehydrate is from 240 to 300 mM. In other embodiments, the molarity ofthe trehalose dehydrate is from 240 to 290 mM. In some embodiments, themolarity of the trehalose dehydrate is from 250 to 290 mM. In otherembodiments, the molarity of the trehalose dehydrate is from 260 to 290mM. In some embodiments, the molarity of the trehalose dehydrate is from270 to 290 mM. In certain embodiments, the molarity of the trehalosedehydrate is from 244 to 245 mM. In other embodiments, the molarity ofthe trehalose dehydrate is about 280 mM.

In one embodiment, the concentration of hydrochloric acid useful in thepharmaceutical compositions provided herein is in the range of from 0.01to 1% (v/v). In another embodiment, the concentration of hydrochloricacid is in the range of from 0.025 to 0.75% (v/v). In anotherembodiment, the concentration of hydrochloric acid is in the range offrom 0.05 to 0.5% (v/v). In another embodiment, the concentration ofhydrochloric acid is in the range of from 0.075 to 0.25% (v/v). In oneparticular embodiment, the concentration of hydrochloric acid is 0.1%(v/v). In certain embodiments, a formulation volume of 1000 mL comprisesfrom 0.8 to 1.1 mL of hydrochloric acid. In a specific embodiment, aformulation volume of 1000 mL comprises from 0.9 to 1.0 mL ofhydrochloric acid. In certain embodiments, a formulation volume of 1000mL comprises 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99or 1.00 mL of hydrochloric acid. In certain embodiments, a formulationvolume of 1000 mL comprises 0.90 mL of hydrochloric acid. In certainembodiments, a formulation volume of 1000 mL comprises 0.91 mL ofhydrochloric acid. In certain embodiments, a formulation volume of 1000mL comprises 0.92 mL of hydrochloric acid. In certain embodiments, aformulation volume of 1000 mL comprises 0.93 mL of hydrochloric acid. Incertain embodiments, a formulation volume of 1000 mL comprises 0.94 mLof hydrochloric acid. In certain embodiments, a formulation volume of1000 mL comprises 0.95 mL of hydrochloric acid. In certain embodiments,a formulation volume of 1000 mL comprises 0.96 mL of hydrochloric acid.In certain embodiments, a formulation volume of 1000 mL comprises 0.97mL of hydrochloric acid. In certain embodiments, a formulation volume of1000 mL comprises 0.98 mL of hydrochloric acid. In certain embodiments,a formulation volume of 1000 mL comprises 0.99 mL of hydrochloric acid.In certain embodiments, a formulation volume of 1000 mL comprises 1.00mL of hydrochloric acid.

In one embodiment, the concentration of TWEEN-20 useful in thepharmaceutical compositions provided herein is in the range of from0.001 to 0.1% (v/v). In another embodiment, the concentration ofTWEEN-20 is in the range of from 0.0025 to 0.075% (v/v). In oneembodiment, the concentration of TWEEN-20 is in the range of from 0.005to 0.05% (v/v). In another embodiment, the concentration of TWEEN-20 isin the range of from 0.0075 to 0.025% (v/v). In one particularembodiment, the concentration of TWEEN-20 is 0.01% (v/v). In certainembodiments, a formulation volume of 1000 mL comprises 0.1 mL TWEEN-20.

In one embodiment, the molarity of potassium phosphate useful in thepharmaceutical compositions provided herein is in the range of between 5and 50 mM. In one embodiment, the molarity of potassium phosphate is inthe range of 10 and 40 mM. In another embodiment, the molarity ofpotassium phosphate is in the range of 10 and 35 mM. In one embodiment,the molarity of potassium phosphate is in the range of 10 and 30 mM. Inanother embodiment, the molarity of potassium phosphate is in the rangeof 10 and 25 mM. In one embodiment, the molarity of potassium phosphateis in the range of 10 and 20 mM. In one particular embodiment, themolarity of potassium phosphate is 10 mM. In one particular embodiment,the molarity of potassium phosphate is 20 mM.

In specific embodiments, the pharmaceutical composition does notcomprise potassium phosphate.

In one embodiment, the molarity of sodium chloride useful in thepharmaceutical compositions provided herein is in the range of between50 and 250 mM. In another embodiment, the molarity of sodium chloride isin the range of 75 and 225 mM. In one embodiment, the molarity of sodiumchloride is in the range of 100 and 200 mM. In another embodiment, themolarity of sodium chloride is in the range of 125 and 175 mM. In oneembodiment, the molarity of sodium chloride is in the range of 130 and160 mM. In another embodiment, the molarity of potassium phosphate is inthe range of 140 and 150 mM. In one particular embodiment, the molarityof sodium chloride is 125 mM. In one particular embodiment, the molarityof sodium chloride is 140 mM. In one particular embodiment, the molarityof sodium chloride is 150 mM.

In specific embodiments, the pharmaceutical composition does notcomprise a salt. In other specific embodiments, the pharmaceuticalcomposition does not comprise sodium chloride.

In one embodiment, the pharmaceutical composition having a pH in a rangeof 6.5 and 9.5. In another embodiment, the pharmaceutical compositionhaving a pH in a range of 6.8 and 9.3. In one embodiment, thepharmaceutical composition having a pH in a range of 7.0 and 9.0. Inanother embodiment, the pharmaceutical composition having a pH in arange of 7.3 and 8.7. In one embodiment, the pharmaceutical compositionhaving a pH in a range of 7.5 and 8.5. In another embodiment, thepharmaceutical composition having a pH in a range of 7.7 and 8.2. In oneparticular embodiment, the pH of the pharmaceutical composition is 7.3.In one particular embodiment, the pH of the pharmaceutical compositionis 7.5. In one particular embodiment, the pH of the pharmaceuticalcomposition is 8.0. In one particular embodiment, the pH of thepharmaceutical composition is 8.5. The measurements of the pH can betaken at the temperature of 4° C.

In a one embodiment, a pharmaceutical composition provided hereincomprises 20 mM Tris (e.g., 2.423 g in 1000 mL solution). In anotherembodiment, a pharmaceutical composition provided herein comprises 8.2%(w/v) trehalose dihydrate (e.g., 92.55 g in 1000 mL solution). Inanother embodiment, a pharmaceutical composition provided hereincomprises 0.01% (v/v) TWEEN-20 (e.g., 0.1 mL in 1000 mL solution). Inyet another embodiment, a pharmaceutical compositing provided hereincomprises hydrochloric acid, N.F. (e.g., 0.94 mL in 1000 mL solution),and, in certain embodiments, further comprises water WFI quality (addedto 999 mL). Any combination of two, three, four, five or all six of theforegoing components are also contemplated. In some embodiments, apharmaceutical composition provided herein has a pH from 7.7 to 8.2,such as above 7.7, above 7.8, above 7.9, above 8.0 or above 8.1, whenmeasured at 25° C. In some embodiments, a pharmaceutical compositionprovided herein has a pH above 7.7, when measured at 25° C. In someembodiments, a pharmaceutical composition provided herein has a pH above7.8, when measured at 25° C. In some embodiments, a pharmaceuticalcomposition provided herein has a pH above 7.9, when measured at 25° C.In some embodiments, a pharmaceutical composition provided herein has apH above 8.0, when measured at 25° C. In some embodiments, apharmaceutical composition provided herein has a pH above 8.1, whenmeasured at 25° C. In one embodiment, a pharmaceutical compositionprovided herein has a pH of from 7.9 to 8.1, for example, when measuredat 25° C. In certain embodiments, a pharmaceutical composition providedherein has a pH of 8.0, for example, when measured at 25° C. In someembodiments, a pharmaceutical composition provided herein has a pH of8.5, for example, when measured at 4° C.

Thus, in one embodiment, the pharmaceutical composition comprisespotassium phosphate, sodium chloride, Tromethamine (Tris), Trehalosedihydrate, TWEEN-20, hydrochloric acid, water, or any combinationthereof. In a specific embodiment, the pharmaceutical compositioncomprises potassium phosphate, sodium chloride, Tromethamine (Tris),Trehalose dihydrate, TWEEN-20, hydrochloric acid, and water.

In a specific embodiment, a pharmaceutical composition provided hereincomprises 20 mM Tris (2.423 g on 1000 mL solution), 8.2% (w/v) trehalosedihydrate (92.55 g in 1000 mL solution), 0.01% (v/v) TWEEN-20 (0.1 mL in1000 mL solution), hydrochloric acid, N.F. (0.94 mL in 1000 mLsolution), and water WFI quality (added to 999 mL), and has a pH of 8.5(measured at 4° C.).

In specific embodiments, a pharmaceutical composition provided hereindoes not comprise salt. In certain embodiments, a pharmaceuticalprovided herein comprises a sugar, such as trehalose. In specificembodiments, a pharmaceutical composition provided herein comprises asugar, but does not comprise a salt. In some embodiments, apharmaceutical composition provided herein has a pH from 7.7 to 8.2,when measured at 25° C.

In some embodiments, the pharmaceutical composition has an osmolalityfrom 250 to 350 Osmol/L. In some embodiments, the pharmaceuticalcomposition has an osmolality from 275 to 325 Osmol/L. In someembodiments, the pharmaceutical composition has an osmolality from 295to 305 Osmol/L. In some embodiments, the pharmaceutical composition hasan osmolality of 295 Osmol/L. In some embodiments, the pharmaceuticalcomposition has an osmolality of 296 Osmol/L. In some embodiments, thepharmaceutical composition has an osmolality of 297 Osmol/L. In someembodiments, the pharmaceutical composition has an osmolality of 298Osmol/L. In some embodiments, the pharmaceutical composition has anosmolality of 299 Osmol/L. In some embodiments, the pharmaceuticalcomposition has an osmolality of 300 Osmol/L. In some embodiments, thepharmaceutical composition has an osmolality of 301 Osmol/L. In someembodiments, the pharmaceutical composition has an osmolality of 302Osmol/L. In some embodiments, the pharmaceutical composition has anosmolality of 303 Osmol/L. In some embodiments, the pharmaceuticalcomposition has an osmolality of 304 Osmol/L. In some embodiments, thepharmaceutical composition has an osmolality of 305 Osmol/L.

Although certain numbers (and numerical ranges thereof) are provided, itis understood that, in certain embodiments, numerical values within,e.g., 2%, 5%, 10%, 15% or 20% of said numbers (or numerical ranges) arealso contemplated. Other exemplary pharmaceutical compositions areprovided in the Experimental section below.

A primary solvent in a vehicle may be either aqueous or non-aqueous innature. In addition, the vehicle may contain other pharmaceuticallyacceptable excipients for modifying or maintaining the pH, osmolarity,viscosity, sterility or stability of the pharmaceutical composition. Incertain embodiments, the pharmaceutically acceptable vehicle is anaqueous buffer. In other embodiments, a vehicle comprises, for example,sodium chloride and/or sodium citrate.

Pharmaceutical compositions provided herein may contain still otherpharmaceutically-acceptable formulation agents for modifying ormaintaining the rate of release of a peptide and/or an additional agent,as described herein. Such formulation agents include those substancesknown to artisans skilled in preparing sustained-release formulations.For further reference pertaining to pharmaceutically and physiologicallyacceptable formulation agents, see, for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, The Merck Index, 12th Ed. (1996, MerckPublishing Group, Whitehouse, N.J.); and Pharmaceutical Principles ofSolid Dosage Forms (1993, Technonic Publishing Co., Inc., Lancaster,Pa.). Additional pharmaceutical compositions appropriate foradministration are known in the art and are applicable in the methodsand compositions provided herein.

A pharmaceutical composition may be stored in a sterile vial as asolution, suspension, gel, emulsion, solid, or dehydrated or lyophilizedpowder. Such compositions may be stored either in a ready to use form, alyophilized form requiring reconstitution prior to use, a liquid formrequiring dilution prior to use, or other acceptable form. In someembodiments, a pharmaceutical composition is provided in a single-usecontainer (e.g., a single-use vial, ampoule, syringe, or autoinjector(similar to, e.g., an EpiPen®)), whereas a multi-use container (e.g., amulti-use vial) is provided in other embodiments. Any drug deliveryapparatus may be used to deliver peptides and the other agents describedherein, including implants (e.g., implantable pumps) and cathetersystems, both of which are known to the skilled artisan. Depotinjections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release peptides and/or otheragents described herein over a defined period of time. Depot injectionsare usually either solid- or oil-based and generally comprise at leastone of the formulation components set forth herein. The skilled artisanis familiar with possible formulations and uses of depot injections. Incertain embodiments, the use of Nano Precision Medical's depot deliverytechnology (Nano Precision Medical; Emeryville, Calif.) is contemplated.The technology utilizes a titania nanotube membrane that produceszero-order release rates of macromolecules, such as protein and peptidetherapeutics. The biocompatible membrane is housed in a small,subcutaneous implant that provides long-term (e.g., up to one year),constant-rate delivery of therapeutic macromolecules. The technology iscurrently being evaluated, e.g., for the delivery of GLP-1 agonists forthe treatment of Type II diabetes.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byroutes including parenteral (e.g., subcutaneous (s.c.), intravenous,intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion),inhalation, intracavity, intracranial, and transdermal (topical). Otherexemplary routes of administration are set forth herein.

Pharmaceutical compositions may be in the form of a sterile injectableaqueous or oleagenous suspension. This suspension may be formulatedusing suitable dispersing or wetting agents and suspending agentsdisclosed herein or known to the skilled artisan. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Acceptable diluents, solvents anddispersion media that may be employed include water, Ringer's solution,isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany,N.J.) or phosphate buffered saline (PBS), ethanol, polyol (e.g.,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. Moreover, fatty acids such as oleic acid find use inthe preparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

In one embodiment, the pharmaceutical compositions provided herein maybe administered parenterally by injection, infusion, or implantation,for local or systemic administration. Parenteral administration, as usedherein, include intravenous, intraarterial, intraperitoneal,intrathecal, intraventricular, intraurethral, intrasternal,intracranial, intramuscular, intrasynovial, and subcutaneousadministration.

In one embodiment, the pharmaceutical compositions provided herein maybe formulated in any dosage forms that are suitable for parenteraladministration, including solutions, suspensions, emulsions, micelles,liposomes, microspheres, nanosystems, and solid forms suitable forsolutions or suspensions in liquid prior to injection. Such dosage formscan be prepared according to conventional methods known to those skilledin the art of pharmaceutical science (see, e.g., Remington, The Scienceand Practice of Pharmacy, supra).

In one embodiment, the pharmaceutical compositions intended forparenteral administration may include one or more pharmaceuticallyacceptable carriers and excipients, including, but not limited to,aqueous vehicles, water-miscible vehicles, non-aqueous vehicles,antimicrobial agents or preservatives against the growth ofmicroorganisms, stabilizers, solubility enhancers, isotonic agents,buffering agents, antioxidants, local anesthetics, suspending anddispersing agents, wetting or emulsifying agents, complexing agents,sequestering or chelating agents, cryoprotectants, lyoprotectants,thickening agents, pH adjusting agents, and inert gases.

In one embodiment, suitable aqueous vehicles include, but are notlimited to, water, saline, physiological saline or phosphate bufferedsaline (PBS), sodium chloride injection, Ringers injection, isotonicdextrose injection, sterile water injection, dextrose and lactatedRingers injection. Non-aqueous vehicles include, but are not limited to,fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil,olive oil, peanut oil, peppermint oil, safflower oil, sesame oil,soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, andmedium-chain triglycerides of coconut oil, and palm seed oil.Water-miscible vehicles include, but are not limited to, ethanol,1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol300 and polyethylene glycol 400), propylene glycol, glycerin,N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

In one embodiment, suitable antimicrobial agents or preservativesinclude, but are not limited to, phenols, cresols, mercurials, benzylalcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates,thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl-and propyl-parabens, and sorbic acid. Suitable isotonic agents include,but are not limited to, sodium chloride, glycerin, and dextrose.Suitable buffering agents include, but are not limited to, phosphate andcitrate. Suitable antioxidants are those as described herein, includingbisulfite and sodium metabisulfite. Suitable local anesthetics include,but are not limited to, procaine hydrochloride. Suitable suspending anddispersing agents are those as described herein, including sodiumcarboxymethylcellulose, hydroxypropyl methylcellulose, andpolyvinylpyrrolidone. Suitable emulsifying agents include thosedescribed herein, including polyoxyethylene sorbitan monolaurate,polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate.Suitable sequestering or chelating agents include, but are not limitedto EDTA. Suitable pH adjusting agents include, but are not limited to,sodium hydroxide, hydrochloric acid, citric acid, and lactic acid.Suitable complexing agents include, but are not limited to,cyclodextrins, including α-cyclodextrin, β-cyclodextrin,hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, andsulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).

In one embodiment, the pharmaceutical compositions provided herein maybe formulated for single or multiple dosage administration. The singledosage formulations are packaged in an ampoule, a vial, or a syringe.The multiple dosage parenteral formulations may contain an antimicrobialagent at bacteriostatic or fungistatic concentrations. All parenteralformulations must be sterile, as known and practiced in the art.

In one embodiment, the pharmaceutical compositions are provided asready-to-use sterile solutions. In another embodiment, thepharmaceutical compositions are provided as sterile dry solubleproducts, including lyophilized powders and hypodermic tablets, to bereconstituted with a vehicle prior to use. In yet another embodiment,the pharmaceutical compositions are provided as ready-to-use sterilesuspensions. In yet another embodiment, the pharmaceutical compositionsare provided as sterile dry insoluble products to be reconstituted witha vehicle prior to use. In still another embodiment, the pharmaceuticalcompositions are provided as ready-to-use sterile emulsions.

In one embodiment, the pharmaceutical compositions provided herein maybe formulated as immediate or modified release dosage forms, includingdelayed-, sustained, pulsed-, controlled, targeted-, andprogrammed-release forms.

In one embodiment, the pharmaceutical compositions may be formulated asa suspension, solid, semi-solid, or thixotropic liquid, foradministration as an implanted depot. In one embodiment, thepharmaceutical compositions provided herein are dispersed in a solidinner matrix, which is surrounded by an outer polymeric membrane that isinsoluble in body fluids but allows the active ingredient in thepharmaceutical compositions diffuse through.

In one embodiment, suitable inner matrixes includepolymethylmethacrylate, polybutyl-methacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethylene terephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers, such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinyl alcohol, andcross-linked partially hydrolyzed polyvinyl acetate.

In one embodiment, suitable outer polymeric membranes includepolyethylene, polypropylene, ethylene/propylene copolymers,ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers,silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinatedpolyethylene, polyvinylchloride, vinyl chloride copolymers with vinylacetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyloxyethanol copolymer,and ethylene/vinyl acetate/vinyl alcohol terpolymer.

Pharmaceutical compositions may be in a form suitable for oral use, forexample, as tablets, capsules, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups, solutions, microbeads or elixirs. Pharmaceuticalcompositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions. Such compositions may contain one or more agents such assweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets containing a peptide provided herein may be inadmixture with non-toxic pharmaceutically acceptable excipients suitablefor the manufacture of tablets. These excipients include, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated by techniques known in the art toform osmotic therapeutic tablets for controlled release. Additionalagents include biodegradable or biocompatible particles or a polymericsubstance such as polyesters, polyamine acids, hydrogel, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinylacetate,methylcellulose, carboxymethylcellulose, protamine sulfate, orlactide/glycolide copolymers, polylactide/glycolide copolymers, orethylenevinylacetate copolymers in order to control delivery of anadministered composition. For example, the oral agent can be entrappedin microcapsules prepared by coacervation techniques or by interfacialpolymerization, by the use of hydroxymethylcellulose orgelatin-microcapsules or poly (methylmethacrolate) microcapsules,respectively, or in a colloid drug delivery system. Colloidal dispersionsystems include macromolecule complexes, nano-capsules, microspheres,microbeads, and lipid-based systems, including oil-in-water emulsions,micelles, mixed micelles, and liposomes. Methods of preparing liposomesare described in, for example, U.S. Pat. Nos. 4,235,871, 4,501,728, and4,837,028. Methods for the preparation of the above-mentionedformulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients aresuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be a naturally-occurring phosphatide, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxy-ethylene stearate, or condensation productsof ethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

Pharmaceutical compositions provided herein may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example, liquidparaffin, or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example, gum acacia or gum tragacanth;naturally-occurring phosphatides, for example, soy bean, lecithin, andesters or partial esters derived from fatty acids; hexitol anhydrides,for example, sorbitan monooleate; and condensation products of partialesters with ethylene oxide, for example, polyoxyethylene sorbitanmonooleate.

Pharmaceutical compositions can also include carriers to protect thecomposition against rapid degradation or elimination from the body, suchas a controlled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed. Prolongedabsorption of injectable pharmaceutical compositions can be achieved byincluding an agent that delays absorption, for example, aluminummonostearate or gelatin. Prevention of the action of microorganisms canbe achieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike.

Also provided herein are peptides and/or one or more additional agentsdescribed herein in the form of suppositories for rectal administration.The suppositories can be prepared by mixing a peptide and/or one or moreadditional agents described herein with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include, but are not limited to, cocoa butter andpolyethylene glycols.

5.3 Peptides

In certain embodiments, the pharmaceutical compositions, formulationsand dosage forms provided herein comprise one or more peptides orpeptide sequences provided herein. In certain embodiments, thepharmaceutical compositions, formulations and dosage forms providedherein comprise one or more variants of FGF19 peptide sequences, fusionsof FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences having one or moreactivities associated with the treatment and/or prevention of a bileacid-related or associated disorder (e.g., PBC), a metabolic disorder ora cancer or tumor. In certain embodiments, the activity is a glucoselowering activity. Such variants and fusions (chimeras) of FGF19 and/orFGF21 peptide sequences include sequences that do not substantiallyincrease or induce HCC formation or HCC tumorigenesis and/or do notinduce a substantial elevation or increase in lipid profile.

In one embodiment, a chimeric peptide sequence includes or consists ofan N-terminal region having at least seven amino acid residues and theN-terminal region having a first amino acid position and a last aminoacid position, where the N-terminal region has a DSSPL (SEQ ID NO:121)or DASPH (SEQ ID NO:122) sequence; and a C-terminal region having aportion of FGF19 and the C-terminal region having a first amino acidposition and a last amino acid position, where the C-terminal regionincludes amino acid residues 16-29 of FGF19 (WGDPIRLRHLYTSG; SEQ IDNO:169) and the W residue corresponds to the first amino acid positionof the C-terminal region. In particular embodiments, the variant is M70:

MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 70).In particular embodiments, the variant is M69:

RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M69) (SEQ ID NO: 69).

In another embodiment, a chimeric peptide sequence includes or consistsof an N-terminal region having a portion of FGF21 and the N-terminalregion having a first amino acid position and a last amino acidposition, where the N-terminal region has a GQV sequence and the Vresidue corresponds to the last amino acid position of the N-terminalregion; and a C-terminal region having a portion of FGF19 and theC-terminal region having a first amino acid position and a last aminoacid position where the C-terminal region includes amino acid residues21-29 of FGF19 (RLRHLYTSG; SEQ ID NO: 185) and the R residue correspondsto the first position of the C-terminal region.

In particular aspects, modifications to the Loop-8 region of FGF19 aredisclosed herein that possess favorable metabolic parameters withoutexhibiting substantial tumorigenicity. Herein, FGF19 residues 127-129are defined as constituting the Loop-8 region, although in theliterature the Loop-8 region is sometimes defined as including orconsisting of other residues (e.g., residues 125-129). Certaincombinations of R127L and P128E substitutions to the FGF19 framework hadan unexpectedly positive effect on HCC formation. Even moresurprisingly, a combination of R127L and P128E substitutions and asubstitution of Gln (Q) for Leu (L) in the FGF19 core region had an evenmore significant effect on preventing HCC formation.

Accordingly, variants of FGF19 Loop-8 region are included since they canreduce or eliminate substantial, measurable or detectable HCC formation.Furthermore, the effect of reducing HCC formation may be enhanced bymodifications to amino acid residues outside of the Loop-8 region (e.g.,substitutions of amino acid residues in the core region, such as theregion corresponding to amino acids 21-29 of SEQ ID NO:99). In someembodiments, the Loop-8 modified variant comprises a substitution in theFGF19 Loop-8 region corresponding to amino acids 127-129 of SEQ IDNO:99. In certain embodiments, the Loop-8 modified variant comprises asubstitution in the FGF19 Loop-8 region corresponding to (i) a R127Lsubstitution, (ii) a P128E substitution, or (iii) a R127L substitutionand a P128E substitution. In some embodiments, the FGF19 variantcomprises or further comprises a substitution in the core regioncorresponding to amino acids 21-29 of SEQ ID NO:99. In certainembodiments, the FGF19 variant comprises or further comprises asubstitution in the core region corresponding to a L22Q substitution. Incertain embodiments, the amino acid sequence of the peptide comprisesone amino acid substitution to the EIRPD (amino acids 2-6 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19. In someembodiments, the amino acid sequence of the peptide comprises two aminoacid substitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190) aminoacid sequence in the Loop-8 region of FGF19. In other embodiments, theamino acid sequence of the peptide comprises three amino acidsubstitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In certain embodiments, theamino acid sequence of the peptide comprises four amino acidsubstitutions to the EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In some embodiments, the aminoacid sequence of the peptide comprises five amino acid substitutions tothe EIRPD (amino acids 2-6 of SEQ ID NO:190) amino acid sequence in theLoop-8 region of FGF19. In certain embodiments, the amino acid sequenceof the peptide comprises one amino acid substitution to the IRP (aminoacids 3-5 of SEQ ID NO:190) amino acid sequence in the Loop-8 region ofFGF19. In some embodiments, the amino acid sequence of the peptidecomprises two amino acid substitutions to the IRP (amino acids 3-5 ofSEQ ID NO:190) amino acid sequence in the Loop-8 region of FGF19. Inother embodiments, the amino acid sequence of the peptide comprisesthree amino acid substitutions to the IRP (amino acids 3-5 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19. In certainembodiments, the amino acid sequence of the peptide comprises one aminoacid substitution to the RP (amino acids 4-5 of SEQ ID NO:190) aminoacid sequence in the Loop-8 region of FGF19. In some embodiments, theamino acid sequence of the peptide comprises two amino acidsubstitutions to the RP (amino acids 4-5 of SEQ ID NO:190) amino acidsequence in the Loop-8 region of FGF19. In certain embodiments, theamino acid substitution to the RP (amino acids 4-5 of SEQ ID NO:190)amino acid sequence in the Loop-8 region of FGF19 is an Arg (R) to Leu(L) substitution. In other embodiments, the substitution to the RP(amino acids 4-5 of SEQ ID NO:190) amino acid sequence in the Loop-8region of FGF19 is a Pro (P) to Glu (E) substitution. In someembodiments, the substitutions to the RP (amino acids 4-5 of SEQ IDNO:190) amino acid sequence in the Loop-8 region of FGF19 is an Arg (R)to Leu (L) substitution and a Pro (P) to Glu (E) substitution. Inspecific embodiments, the foregoing substitution(s) in the Loop-8 regionof FGF19 is in the corresponding FGF19 sequence thereof in a variantpeptide provided herein. That is, said substitutions within acorresponding FGF19 sequence (e.g., EIRPD, IRP or RP) of a peptidevariant provided herein is also contemplated.

In some embodiments, the Loop-8 modified variant is M70:

MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 70),comprising a substitution in the FGF19 Loop-8 region (underlined). Incertain embodiments, the Loop-8 modified M70 variant comprises asubstitution in the FGF19 Loop-8 region (underlined) corresponding to(i) a R127L substitution, (ii) a P128E substitution, or (iii) a R127Lsubstitution and a P128E substitution (SEQ. ID NO:204). In certainembodiments, the Loop-8 modified M70 variant further comprises orfurther comprises a substitution in the FGF19 core region. In someembodiments, the Loop-8 modified M70 variant comprises a L18Qsubstitution.

In some embodiments, the Loop-8 modified variant is M69:

RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M69) (SEQ ID NO: 69),comprising a substitution in the FGF19 Loop-8 region (underlined). Incertain embodiments, the Loop-8 modified M69 variant comprises asubstitution in the FGF19 Loop-8 region (underlined) corresponding to(i) a R100L substitution, (ii) a P101E substitution, or (iii) a R100Lsubstitution and a P101E substitution. In certain embodiments, theLoop-8 modified M69 variant further comprises or further comprises asubstitution in the FGF19 core region. In some embodiments, the Loop-8modified M69 variant comprises a L17Q substitution.

In some embodiments, the Loop-8 modified variant comprises asubstitution in the FGF19 Loop-8 region corresponding to amino acids127-129 of SEQ ID NO:3. In certain embodiments, the Loop-8 modifiedvariant comprises a substitution in the FGF19 Loop-8 regioncorresponding to (i) a R127L substitution, (ii) a P128E substitution, or(iii) a R127L substitution and a P128E substitution. In someembodiments, the FGF19 variant comprises or further comprises asubstitution in the core region corresponding to amino acids 21-29 ofSEQ ID NO:3. In certain embodiments, the FGF19 variant comprises orfurther comprises a substitution in the core region corresponding to aL22Q substitution.

In further embodiments, a peptide sequence includes or consists of aFGF19 variant having one or more amino acid substitutions, insertions ordeletions compared to a reference or wild type FGF19. In additionalembodiments, a peptide sequence includes or consists of a FGF21 sequencevariant having one or more amino acid substitutions, insertions ordeletions compared to a reference or wild type FGF21. In yet additionalembodiments, a peptide sequence includes or consists of a portion of aFGF19 sequence fused to a portion of a FGF21 sequence. In stilladditional embodiments, a peptide sequence includes or consists of aportion of a FGF19 sequence fused to a portion of a FGF21 sequence,where the FGF19 and/or FGF21 sequence portion(s) have one or more aminoacid substitutions, insertions or deletions compared to a reference orwild type FGF19 and/or FGF21. Examples of such sequences are disclosedin PCT Pub. No. WO 2013/006486 and US Pub. No. 2013/0023474, as well asPCT Publ. No. WO 2014/085365, published Jun. 5, 2014. Table 1 and theSequence Listing also sets forth representative sequences that may beused in the methods provided herein.

In some embodiments, the treatment peptides provided herein includevariants and fusions of FGF19 and/or FGF21 peptide sequences. In oneembodiment, the treatment peptides include one or more variant or fusionFGF19 and/or FGF21 peptide. In other embodiments, the methods providedherein include contacting or administering to a subject one or morenucleic acid molecules encoding a variant or fusion FGF19 and/or FGF21peptide sequence (for example, an expression control element in operablelinkage with the nucleic acid encoding the peptide sequence, optionallyincluding a vector), in an amount effective for treating a bileacid-related or associated disorder.

A representative reference or wild type FGF19 sequence is set forth as:

RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVR SPSFEK (SEQ ID NO: 99).

A representative reference or wild type FGF21 sequence is set forth as:

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 100).FGF21 allelic variants include, e.g., M70, M71 and M72.

The terms “peptide,” “protein,” and “polypeptide” sequence are usedinterchangeably herein to refer to two or more amino acids, or“residues,” including chemical modifications and derivatives of aminoacids, covalently linked by an amide bond or equivalent. The amino acidsforming all or a part of a peptide may be from among the known 21naturally occurring amino acids, which are referred to by both theirsingle letter abbreviation or common three-letter abbreviation. In thepeptide sequences provided herein, conventional amino acid residues havetheir conventional meaning. Thus, “Leu” is leucine, “Ile” is isoleucine,“Nle” is norleucine, and so on.

In various particular aspects, a peptide or chimeric sequence providedherein has at the N-terminal region first amino acid position an “M”residue, an “R” residue, a “S” residue, a “H” residue, a “P” residue, a“L” residue or an “D” residue. In various alternative particularaspects, a peptide or chimeric sequence peptide sequence does not have a“M” residue or an “R” residue at the first amino acid position of theN-terminal region.

Also provided herein are subsequences, variants and modified forms ofthe exemplified peptide sequences (including the FGF19 and FGF21variants and subsequences listed in the Sequence Listing, or Table 1),so long as the foregoing retains at least a detectable or measurableactivity or function. Also, certain exemplified variant peptides, forexample, those having all or a portion of FGF21 sequence at theamino-terminus, have an “R” residue positioned at the N-terminus, whichcan be omitted. Similarly, certain exemplified variant peptides, includean “M” residue positioned at the N-terminus, which can be appended to orfurther substituted for an omitted residue, such as an “R” residue. Moreparticularly, in various embodiments peptide sequences at the N-terminusinclude any of: RDSS (SEQ ID NO:115), DSS, MDSS (SEQ ID NO:116) or MRDSS(SEQ ID NO:117). Furthermore, when a “M” residue is adjacent to a “S”residue, the “M” residue may be cleaved such that the “M” residue isdeleted from the peptide sequence, whereas when the “M” residue isadjacent to a “D” residue, the “M” residue may not be cleaved. Thus, byway of example, in various embodiments peptide sequences include thosewith the following residues at the N-terminus: MDSSPL (SEQ ID NO:119),MSDSSPL (SEQ ID NO:120) (cleaved to SDSSPL (SEQ ID NO:112)) and MSSPL(SEQ ID NO:113) (cleaved to SSPL (SEQ ID NO:114)).

Exemplified herein are peptide sequences, distinct from reference FGF19and FGF21 polypeptides set forth herein, that modulate bile acidhomeostasis, hyperglycemic conditions, insulin resistance,hyperinsulinemia, glucose intolerance, metabolic syndrome, or relateddisorders, in vivo (e.g., Table 1 and the Sequence Listing).Non-limiting particular examples are a peptide sequence withamino-terminal amino acids 1-16 of FGF21 fused to carboxy-terminal aminoacids 21-194 of FGF19; a peptide sequence with amino-terminal aminoacids 1-147 of FGF19 fused to carboxy-terminal amino acids 147-181 ofFGF21; a peptide sequence with amino-terminal amino acids 1-20 of FGF19fused to carboxy-terminal amino acids 17-181 of FGF21; a peptidesequence with amino-terminal amino acids 1-146 of FGF21 fused tocarboxy-terminal amino acids 148-194 of FGF19; and a peptide sequencewith amino-terminal amino acids 1-20 of FGF19 fused to internal aminoacids 17-146 of FGF21 fused to carboxy-terminal amino acids 148-194 ofFGF19.

Additional particular peptides sequences have a WGDPI (SEQ ID NO:170)sequence motif corresponding to the WGDPI sequence of amino acids 16-20of FGF19 (SEQ ID NO:99), lack a WGDPI (SEQ ID NO:170) sequence motifcorresponding to the WGDPI sequence of amino acids 16-20 of FGF19 (SEQID NO:99), or have a substituted (i.e., mutated) WGDPI (SEQ ID NO:170)sequence motif corresponding to FGF19 WGDPI sequence of amino acids16-20 of FGF19 (SEQ ID NO:99).

Particular peptide sequences provided herein also include sequencesdistinct from FGF19 and FGF21 (e.g., as set forth herein), and FGF 19variant sequences having any GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for FGF19 WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20. Accordingly, the wild-type FGF19 and FGF21 (e.g., as setforth herein as SEQ ID NOS:99 and 100, respectively) may be excludedsequences, and FGF19 having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19 may also be excluded. This exclusion, however, doesnot apply to where a sequence has, for example, 3 FGF21 residues fusedto FGF19 having, for example, any of GQV, GQV, GDI, or GPI, or 2 FGF21residues fused to any of WGPI (SEQ ID NO:171), WGDI (SEQ ID NO:173),GDPI (SEQ ID NO:174), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), orWGDP (SEQ ID NO:183).

Particular non-limiting examples of peptide sequences include or consistof all or a part of a sequence variant specified herein as M1-M98 (SEQID NOs:1-52, 192, and 54-98, respectively). More particular non-limitingexamples of peptide sequences include or consist of all or a part of asequence set forth as:

HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M5-R) (SEQ ID NO: 160)(FGF21 sequences can also include an “R” residue at the amino terminus);

DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 138 and 161);RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M1) (SEQ ID NO: 1 or 139);RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M2) (SEQ ID NO: 2 or 140);DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 141);RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M69) (SEQ ID NO: 69);RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M52) (SEQ ID NO: 52);HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M5-R) (SEQ ID NO: 160);HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71) (SEQ ID NO: 71);HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72) (SEQ ID NO: 72);HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73) (SEQ ID NO: 73);RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M3) (SEQ ID NO: 3);RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK(M48) (SEQ ID NO: 48, 6 or 148);RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M49) (SEQ ID NO: 49, 7 or 149);RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M50) (SEQ ID NO: 50);RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M51) (SEQ ID NO: 51, 36 or 155);MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M53) (SEQ ID NO: 192);MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70) (SEQ ID NO: 70);RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M139) (SEQ ID NO: 193);RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140) (SEQ ID NO: 194);RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141) (SEQ ID NO: 195); orRPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160) (SEQ ID NO: 196);or a subsequence or fragment thereof any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the R terminal residue is deleted.

Additional particular non-limiting examples of peptide sequences, havingat the N-terminus, a peptide sequence including or consisting of all ora part of any of:

HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO: 160);DSSPLLQFGGQVRLRHLYTSG (M6) (M6-R) (amino acids 2-22 of SEQ ID NO: 6);RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID NO: 7);HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID NO: 8);HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ ID NO: 9);HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of SEQ ID NO: 10);RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27 of SEQ ID NO: 11);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R) (amino acids 2-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15) (amino acids 1-27 of SEQ ID NO: 15);RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO: 16);RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID NO: 17);RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ ID NO: 18);RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of SEQ ID NO: 19);RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27 of SEQ ID NO: 20);RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids 1-27 of SEQ ID NO: 21);RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino acids 1-27 of SEQ ID NO: 22);RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23) (amino acids 1-27 of SEQ ID NO: 23);RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24) (amino acids 1-27 of SEQ ID NO: 24);RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID NO: 25);RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ ID NO: 26);RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of SEQ ID NO: 27);RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26 of SEQ ID NO: 28);RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids 1-28 of SEQ ID NO: 29);RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino acids 1-29 of SEQ ID NO: 30);RHPIPDSSPLLQFGAQVRLRHLYTSG (M31) (amino acids 1-26 of SEQ ID NO: 31);RHPIPDSSPLLQFGDQVRLRHLYTSG (M32) (amino acids 1-26 of SEQ ID NO: 32);RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID NO: 33);RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ ID NO: 34);RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of SEQ ID NO: 35);RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26 of SEQ ID NO: 36);RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids 1-26 of SEQ ID NO: 37);RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino acids 1-26 of SEQ ID NO: 38);RHPIPDSSPLLQFGGQTRLRHLYTSG (M39) (amino acids 1-26 of SEQ ID NO: 39);RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40) (amino acids 1-28 of SEQ ID NO: 40);DAGPHVHYGWGDPIRLRHLYTSG (M74-R) (amino acids 2-24 of SEQ ID NO: 74);VHYGWGDPIRLRHLYTSG (M75-R) (amino acids 2-19 of SEQ ID NO: 75);RLRHLYTSG (M77-R) (amino acids 2-10 of SEQ ID NO: 77);RHPIPDSSPLLQFGWGDPIRLRHLYTSG (M9) (amino acids 1-28 of SEQ ID NO: 9);RHPIPDSSPLLQWGDPIRLRHLYTSG (M8) (amino acids 1-26 of SEQ ID NO: 8);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID NO: 10);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27 of SEQ ID NO: 43); orRDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22 of SEQ ID NO: 6);and for any of the foregoing peptide sequences the amino terminal Rresidue may be deleted.

Peptide sequences provided herein additionally include those withreduced or absent induction or formation of HCC compared to FGF19, or aFGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID NO:170) sequenceat amino acids 16-20 of FGF19. Peptide sequences provided herein alsoinclude those with greater glucose lowering activity compared to FGF19,or a FGF 19 variant sequence having any of GQV, GDI, WGPI, WGPI (SEQ IDNO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19. Peptide sequencesprovided herein moreover include those with less lipid (e.g.,triglyceride, cholesterol, non-HDL or HDL) increasing activity comparedto FGF19, or a FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19.

Typically, the number of amino acids or residues in a peptide sequenceprovided herein will total less than about 250 (e.g., amino acids ormimetics thereof). In various particular embodiments, the number ofresidues comprise from about 20 up to about 200 residues (e.g., aminoacids or mimetics thereof). In additional embodiments, the number ofresidues comprise from about 50 up to about 200 residues (e.g., aminoacids or mimetics thereof). In further embodiments, the number ofresidues comprise from about 100 up to about 195 residues (e.g., aminoacids or mimetics thereof) in length.

Amino acids or residues can be linked by amide or by non-natural andnon-amide chemical bonds including, for example, those formed withglutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, orN,N′-dicyclohexylcarbodiimide (DCC). Non-amide bonds include, forexample, ketomethylene, aminomethylene, olefin, ether, thioether and thelike (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357 (1983), “Peptide and BackboneModifications,” Marcel Decker, NY). Thus, when a peptide provided hereinincludes a portion of a FGF19 sequence and a portion of a FGF21sequence, the two portions need not be joined to each other by an amidebond, but can be joined by any other chemical moiety or conjugatedtogether via a linker moiety.

In some embodiments, the treatment peptides provided herein also includesubsequences, variants and modified forms of the exemplified peptidesequences (including the FGF19 and FGF21 variants and subsequenceslisted in Table 1 and Sequence Listing), so long as the foregoingretains at least a detectable or measurable activity or function. Forexample, certain exemplified variant peptides have FGF19 C-terminalsequence,

PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188)at the C-terminal portion, e.g., following the “TSG” amino acid residuesof the variant.

Also, certain exemplified variant peptides, for example, those havingall or a portion of FGF21 sequence at the amino-terminus, have an “R”residue positioned at the N-terminus, which can be omitted. Similarly,certain exemplified variant peptides, include an “M” residue positionedat the N-terminus, which can be appended to or further substituted foran omitted residue, such as an “R” residue. More particularly, invarious embodiments peptide sequences at the N-terminus include any of:RDSS (SEQ ID NO:115), DSS, MDSS (SEQ ID NO:116) or MRDSS (SEQ IDNO:117). Furthermore, in cells when a “M” residue is adjacent to a “S”residue, the “M” residue may be cleaved such that the “M” residue isdeleted from the peptide sequence, whereas when the “M” residue isadjacent to a “D” residue, the “M” residue may not be cleaved. Thus, byway of example, in various embodiments peptide sequences include thosewith the following residues at the N-terminus: MDSSPL (SEQ ID NO:119),MSDSSPL (SEQ ID NO:120) (cleaved to SDSSPL (SEQ ID NO:112)) and MSSPL(SEQ ID NO:113) (cleaved to SSPL (SEQ ID NO:114)).

Accordingly, in some embodiments, the “peptide,” “polypeptide,” and“protein” sequences provided herein include subsequences, variants andmodified forms of the FGF19 and FGF21 variants and subsequences listedin Table 1 and Sequence Listing, and the FGF19/FGF21 fusions andchimeras listed in Table 1 and Sequence Listing, so long as thesubsequence, variant or modified form (e.g., fusion or chimera) retainsat least a detectable activity or function, e.g., glucose loweringactivity and/or modulation of bile acid homeostasis.

As used herein, the term “modify” and grammatical variations thereof,means that the composition deviates relative to a reference composition,such as a peptide sequence. Such modified peptide sequences, nucleicacids and other compositions may have greater or less activity orfunction, or have a distinct function or activity compared with areference unmodified peptide sequence, nucleic acid, or othercomposition, or may have a property desirable in a protein formulatedfor therapy (e.g. serum half-life), to elicit antibody for use in adetection assay, and/or for protein purification. For example, a peptidesequence provided herein can be modified to increase serum half-life, toincrease in vitro and/or in vivo stability of the protein, etc.

Particular examples of such subsequences, variants and modified forms ofthe peptide sequences exemplified herein (e.g., a peptide sequencelisted in the Sequence Listing or Table 1) include substitutions,deletions and/or insertions/additions of one or more amino acids, to orfrom the amino-terminus, the carboxy-terminus or internally. One exampleis a substitution of an amino acid residue for another amino acidresidue within the peptide sequence. Another is a deletion of one ormore amino acid residues from the peptide sequence, or an insertion oraddition of one or more amino acid residues into the peptide sequence.

The number of residues substituted, deleted or inserted/added are one ormore amino acids (e.g., 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250,or more) of a peptide sequence. Thus, a FGF19 or FGF21 sequence can havefew or many amino acids substituted, deleted or inserted/added (e.g.,1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170,170-180, 180-190, 190-200, 200-225, 225-250, or more). In addition, aFGF19 amino acid sequence can include or consist of an amino acidsequence of about 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60,60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250,or more amino acids from FGF21; or a FGF21 amino acid or sequence caninclude or consist of an amino acid sequence of about 1-3, 3-5, 5-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-225, 225-250, or more amino acids from FGF19.

Specific examples of substitutions include substituting a D residue foran L-residue. Accordingly, although residues are listed in the L-isomerconfiguration, D-amino acids at any particular or all positions of thepeptide sequences provided herein are included, unless a D-isomer leadsto a sequence that has no detectable or measurable function.

Additional specific examples are non-conservative and conservativesubstitutions. A “conservative substitution” is a replacement of oneamino acid by a biologically, chemically or structurally similarresidue. Biologically similar means that the substitution is compatiblewith a biological activity, e.g., activity that improves PBC and/or themanifestations thereof. Structurally similar means that the amino acidshave side chains with similar length, such as alanine, glycine andserine, or having similar size, or the structure of a first, second oradditional peptide sequence is maintained. Chemical similarity meansthat the residues have the same charge or are both hydrophilic andhydrophobic. Particular examples include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methionine,for another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, serine for threonine, etc. Routine assayscan be used to determine whether a subsequence, variant or modified formhas activity, e.g., activity that improves PBC and/or the manifestationsthereof.

Particular examples of subsequences, variants and modified forms of thepeptide sequences exemplified herein have 50%-60%, 60%-70%, 70%-75%,75%-80%, 80%-85%, 85%-90%, 90%-95%, or 96%, 97%, 98%, or 99% identity toa reference peptide sequence. The term “identity” and “homology” andgrammatical variations thereof mean that two or more referenced entitiesare the same. Thus, where two amino acid sequences are identical, theyhave the identical amino acid sequence. “Areas, regions or domains ofidentity” mean that a portion of two or more referenced entities are thesame. Thus, where two amino acid sequences are identical or homologousover one or more sequence regions, they share identity in those regions.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm known in the art. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region. Forexample, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschulet al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI)has exemplary search parameters as follows: Mismatch-2; gap open 5; gapextension 2. For peptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

In the peptide sequences, including subsequences, variants and modifiedforms of the peptide sequences exemplified herein, an “amino acid” or“residue” includes conventional alpha-amino acids as well as beta-aminoacids; alpha, alpha disubstituted amino acids; and N-substituted aminoacids, wherein at least one side chain is an amino acid side chainmoiety as defined herein. An “amino acid” further includes N-alkylalpha-amino acids, wherein the N-terminus amino group has a C₁ to C₆linear or branched alkyl substituent. The term “amino acid” thereforeincludes stereoisomers and modifications of naturally occurring proteinamino acids, non-protein amino acids, post-translationally modifiedamino acids (e.g., by glycosylation, phosphorylation, ester or amidecleavage, etc.), enzymatically modified or synthesized amino acids,derivatized amino acids, constructs or structures designed to mimicamino acids, amino acids with a side chain moiety modified, derivatizedfrom naturally occurring moieties, or synthetic, or not naturallyoccurring, etc. Modified and unusual amino acids are included in thepeptide sequences provided herein (see, for example, in SyntheticPeptides: A User's Guide; Hruby et al., Biochem. J. 268:249 (1990); andToniolo C., Int. J. Peptide Protein Res. 35:287 (1990)).

In addition, protecting and modifying groups of amino acids areincluded. The term “amino acid side chain moiety” as used hereinincludes any side chain of any amino acid, as the term “amino acid” isdefined herein. This therefore includes the side chain moiety innaturally occurring amino acids. It further includes side chain moietiesin modified naturally occurring amino acids as set forth herein andknown to one of skill in the art, such as side chain moieties instereoisomers and modifications of naturally occurring protein aminoacids, non-protein amino acids, post-translationally modified aminoacids, enzymatically modified or synthesized amino acids, derivatizedamino acids, constructs or structures designed to mimic amino acids,etc. For example, the side chain moiety of any amino acid disclosedherein or known to one of skill in the art is included within thedefinition.

A “derivative of an amino acid side chain moiety” is included within thedefinition of an amino acid side chain moiety. Non-limiting examples ofderivatized amino acid side chain moieties include, for example: (a)adding one or more saturated or unsaturated carbon atoms to an existingalkyl, aryl, or aralkyl chain; (b) substituting a carbon in the sidechain with another atom, such as oxygen or nitrogen; (c) adding aterminal group to a carbon atom of the side chain, including methyl(—CH₃), methoxy (—OCH₃), nitro (—NO₂), hydroxyl (—OH), or cyano (—C═N);(d) for side chain moieties including a hydroxy, thiol or amino groups,adding a suitable hydroxy, thiol or amino protecting group; or (e) forside chain moieties including a ring structure, adding one or more ringsubstituents, including hydroxyl, halogen, alkyl, or aryl groupsattached directly or through, e.g., an ether linkage. For amino groups,suitable protecting groups are known to the skilled artisan. Providedsuch derivatization provides a desired activity in the final peptidesequence (e.g., activity that improves PBC and/or the manifestationsthereof).

An “amino acid side chain moiety” includes all such derivatization, andparticular non-limiting examples include: gamma-amino butyric acid,12-amino dodecanoic acid, alpha-aminoisobutyric acid, 6-amino hexanoicacid, 4-(aminomethyl)-cyclohexane carboxylic acid, 8-amino octanoicacid, biphenylalanine, Boc-t-butoxycarbonyl, benzyl, benzoyl,citrulline, diaminobutyric acid, pyrrollysine, diaminopropionic acid,3,3-diphenylalanine, orthonine, citrulline,1,3-dihydro-2H-isoindolecarboxylic acid, ethyl,Fmoc—fluorenylmethoxycarbonyl, heptanoyl (CH₃—(CH₂)₅—C(═O)—), hexanoyl(CH₃—(CH₂)₄—C(═O)—), homoarginine, homocysteine, homolysine,homophenylalanine, homoserine, methyl, methionine sulfoxide, methioninesulfone, norvaline (NVA), phenylglycine, propyl, isopropyl, sarcosine(SAR), tert-butylalanine, and benzyloxycarbonyl.

A single amino acid, including stereoisomers and modifications ofnaturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically-synthesizedamino acids, non-naturally occurring amino acids including derivatizedamino acids, an alpha, alpha disubstituted amino acid derived from anyof the foregoing (i.e., an alpha, alpha disubstituted amino acid,wherein at least one side chain is the same as that of the residue fromwhich it is derived), a beta-amino acid derived from any of theforegoing (i.e., a beta-amino acid which, other than for the presence ofa beta-carbon, is the same as the residue from which it is derived)etc., including all of the foregoing can be referred to herein as a“residue.” Suitable substituents, in addition to the side chain moietyof the alpha-amino acid, include C₁ to C₆ linear or branched alkyl. Aibis an example of an alpha, alpha disubstituted amino acid. While alpha,alpha disubstituted amino acids can be referred to using conventional L-and D-isomeric references, it is to be understood that such referencesare for convenience, and that where the substituents at thealpha-position are different, such amino acid can interchangeably bereferred to as an alpha, alpha disubstituted amino acid derived from theL- or D-isomer, as appropriate, of a residue with the designated aminoacid side chain moiety. Thus (S)-2-Amino-2-methyl-hexanoic acid can bereferred to as either an alpha, alpha disubstituted amino acid derivedfrom L-Nle (norleucine) or as an alpha, alpha disubstituted amino acidderived from D-Ala. Similarly, Aib can be referred to as an alpha, alphadisubstituted amino acid derived from Ala. Whenever an alpha, alphadisubstituted amino acid is provided, it is to be understood asincluding all (R) and (S) configurations thereof.

An “N-substituted amino acid” includes any amino acid wherein an aminoacid side chain moiety is covalently bonded to the backbone amino group,optionally where there are no substituents other than H in thealpha-carbon position. Sarcosine is an example of an N-substituted aminoacid. By way of example, sarcosine can be referred to as anN-substituted amino acid derivative of Ala, in that the amino acid sidechain moiety of sarcosine and Ala is the same, i.e., methyl.

In certain embodiments, covalent modifications of the peptide sequences,including subsequences, variants and modified forms of the peptidesequences exemplified herein are provided. An exemplary type of covalentmodification includes reacting targeted amino acid residues with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of the peptide.Derivatization with bifunctional agents is useful, for instance, forcross-linking peptide to a water-insoluble support matrix or surface foruse in the method for purifying anti-peptide antibodies, and vice-versa.Commonly used cross linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio] propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, amidation of any C-terminal carboxyl group, etc.

Exemplified peptide sequences, and subsequences, variants and modifiedforms of the peptide sequences exemplified herein can also includealterations of the backbone for stability, derivatives, andpeptidomimetics. The term “peptidomimetic” includes a molecule that is amimic of a residue (referred to as a “mimetic”), including but notlimited to piperazine core molecules, keto-piperazine core molecules anddiazepine core molecules. Unless otherwise specified, an amino acidmimetic of a peptide sequence provided herein includes both a carboxylgroup and amino group, and a group corresponding to an amino acid sidechain, or in the case of a mimetic of Glycine, no side chain other thanhydrogen.

By way of example, these would include compounds that mimic the sterics,surface charge distribution, polarity, etc. of a naturally occurringamino acid, but need not be an amino acid, which would impart stabilityin the biological system. For example, Proline may be substituted byother lactams or lactones of suitable size and substitution; Leucine maybe substituted by an alkyl ketone, N-substituted amide, as well asvariations in amino acid side chain length using alkyl, alkenyl or othersubstituents, others may be apparent to the skilled artisan. Theessential element of making such substitutions is to provide a moleculeof roughly the same size and charge and configuration as the residueused to design the molecule. Refinement of these modifications will bemade by analyzing the compounds in a functional (e.g., glucose lowering)or other assay, and comparing the structure-activity relationship. Suchmethods are within the scope of the skilled artisan working in medicinalchemistry and drug development.

The term “bind,” or “binding,” when used in reference to a peptidesequence, means that the peptide sequence interacts at the molecularlevel. Specific and selective binding can be distinguished fromnon-specific binding using assays known in the art (e.g., competitionbinding, immunoprecipitation, ELISA, flow cytometry, Western blotting).

Peptides and peptidomimetics can be produced and isolated using methodsknown in the art. Peptides can be synthesized, in whole or in part,using chemical methods (see, e.g., Caruthers (1980). Nucleic Acids Res.Symp. Ser. 215; Horn (1980); and Banga, A. K., Therapeutic Peptides andProteins, Formulation, Processing and Delivery Systems (1995) TechnomicPublishing Co., Lancaster, Pa.). Peptide synthesis can be performedusing various solid-phase techniques (see, e.g., Roberge Science 269:202(1995); Merrifield, Methods Enzymol. 289:3 (1997)) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the manufacturer's instructions.Peptides and peptide mimetics can also be synthesized usingcombinatorial methodologies. Synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies known in the art (see, e.g., Organic SynthesesCollective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY).Modified peptides can be produced by chemical modification methods (see,for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, FreeRadic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886(1994)). Peptide sequence variations, derivatives, substitutions andmodifications can also be made using methods such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR-based mutagenesis. Site-directed mutagenesis (Carter et al.,Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315(1985)), restriction selection mutagenesis (Wells et al., Philos. Trans.R. Soc. London SerA 317:415 (1986)) and other techniques can beperformed on cloned DNA to produce peptide sequences, variants, fusionsand chimeras provided herein, and variations, derivatives, substitutionsand modifications thereof.

A “synthesized” or “manufactured” peptide sequence is a peptide made byany method involving manipulation by the hand of man. Such methodsinclude, but are not limited to, the aforementioned, such as chemicalsynthesis, recombinant DNA technology, biochemical or enzymaticfragmentation of larger molecules, and combinations of the foregoing.

Peptide sequences provided herein including subsequences, sequencevariants and modified forms of the exemplified peptide sequences (e.g.,sequences listed in the Sequence Listing or Table 1), can also bemodified to form a chimeric molecule. In certain embodiments, providedherein are peptide sequences that include a heterologous domain. Suchdomains can be added to the amino-terminus or at the carboxyl-terminusof the peptide sequence. Heterologous domains can also be positionedwithin the peptide sequence, and/or alternatively flanked by FGF19and/or FGF21 derived amino acid sequences.

The term “peptide” also includes dimers or multimers (oligomers) ofpeptides. In certain embodiments, dimers or multimers (oligomers) of theexemplified peptide sequences are provided herein, as well assubsequences, variants and modified forms of the exemplified peptidesequences, including sequences listed in the Sequence Listing or Table1.

In certain embodiments, a peptide sequence provided herein comprises anamino acid sequence set forth in Table 1. In other embodiments, apeptide sequence provided herein consists of an amino acid sequence setforth in Table 1.

TABLE 1 SEQ ID NO. Amino Acid Sequence 1.RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 2.RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 3.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 4.RPLAFSDAGPHVHYAWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 5.RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 6.RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 7.RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 8.RHPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 9.RHPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 10.RHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 11.RPLAFSDAGPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 12.RPLAFSDAGPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 13.RPLAFSDAGPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 14.RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 15.RPLAFSDAGPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 16.RPLAFSDAGPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 17.RPLAFSDAGPHVGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 18.RPLAFSDAGPHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 19.RPLAFSDAGPVYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 20.RPLAFSDAGPVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 21.RPLAFSDAGPVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 22.RPLAFSDAGPHVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 23.RPLAFSDAGPHHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 24.RPLAFSDAGPHHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 25.RPLAFSDAGPHVYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 26.RPLAFSDSSPLVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 27.RPLAFSDSSPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 28.RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 29.RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 30.RPLAFSDAGPHVHYAWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 31.RHPIPDSSPLLQFGAQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 32.RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 33.RHPIPDSSPLLQFGPQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 34.RHPIPDSSPLLQFGGAVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 35.RHPIPDSSPLLQFGGEVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 36.RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 37.RHPIPDSSPLLQFGGQARLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 38.RHPIPDSSPLLQFGGQIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 39.RHPIPDSSPLLQFGGQTRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 40.RHPIPDSSPLLQFGWGQPVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 41.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS 42.HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS 43.RPLAFSDAGPHVHYGGDIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 44.RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS 45.HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 46.RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 47.HPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 48.RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 49.RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 50.RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 51.RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 52.RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 53.MDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 54.RPLAFSDAGPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 55.RPLAFSDAGPHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 56.RPLAFSDAGPVYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 57.RPLAFSDAGPVHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 58.RPLAFSDAGPVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 59.RPLAFSDAGPHHGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 60.RPLAFSDAGPHHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 61.RPLAFSDAGPHVGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 62.RPLAFSDAGPHVYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 63.RPLAFSDAGPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 64.RPLAFSDSSPLVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 65.RPLAFSDSSPHVHWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 66.RPLAFSDAGPHLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 67.RPLAFSDAGPHVWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 68.RPLAFSDAGPHVHYWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 69.RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 70.MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 71.HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS 72.HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS 73.HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPV WDGITGE 74.RDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 75.RVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 76.RGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 77.RRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 78.RAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 79.RGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 80.RPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 81.RHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 82.RPLAFSAAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 83.RPLAFSDAAPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 84.RPLAFSDAGAHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 85.RPLAFSDAGPHVHYGAGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 86.RPLAFSDAGPHVHYGWGAPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 87.RPLAFSDAGPHVHYGWGDAICARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPF GLVTGLEAVRSPSFEK 88.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 89.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 90.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 91.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 92.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 93.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQAQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 94.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 95.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 96.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 97.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 98.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 138.DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 139.RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 140.RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 141.DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 142.RHPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 143.RHPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 144.RPLAFSDAGPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 145.RHPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 146.RPLAFSDAGPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 147.RHPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 148.RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 149.RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 150.RHPIPDSSPLLQFGAQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 151.RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 152.RHPIPDSSPLLQFGPQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 153.RHPIPDSSPLLQFGGAVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 154.RHPIPDSSPLLQFGGEVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 155.RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 156.RHPIPDSSPLLQFGGQARLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 157.RHPIPDSSPLLQFGGQIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 158.RHPIPDSSPLLQFGGQTRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 159.RHPIPDSSPLLQFGWGQPVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 160.HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 161.DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 162.HPIPDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 163.HPIPDSSPLLQFGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 164.HPIPDSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 165.HPIPDSSPHVHYGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 166.DAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 167.VHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 168.RLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 188.PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLV TGLEAVRSPSFEK 192.MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 193.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 194.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 195.RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 196.RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 197.RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 198.RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 199.RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 200.RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 201.RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 202.RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 203.RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 204.MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK

In one embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:1. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:2. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:3. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:4. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:5. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:6. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:7. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:8. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:9. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:10. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:11. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:12. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:13. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:14. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:15. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:16. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:17. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:18. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:19. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:20. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:21. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:22. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:23. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:24. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:25. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:26. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:27. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:28. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:29. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:30. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:31. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:32. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:33. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:34. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:35. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:36. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:37. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:38. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:39. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:40. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:41. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:42. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:43. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:44. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:45. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:46. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:47. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:48. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:49. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:50. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:51. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:52. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:53. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:54. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:55. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:56. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:57. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:58. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:59. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:60. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:61. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:62. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:63. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:64. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:65. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:66. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:67. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:68. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:69. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:70. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:71. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:72. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:73. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:74. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:75. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:76. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:77. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:78. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:79. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:80. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:81. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:82. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:83. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:84. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:85. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:86. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:87. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:88. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:89. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:90. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:91. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:92. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:93. In one embodiment, the peptide sequence comprisesan amino acid sequence set forth in SEQ ID NO:94. In another embodiment,the peptide sequence comprises an amino acid sequence set forth in SEQID NO:95. In other embodiments, the peptide sequence comprises an aminoacid sequence set forth in SEQ ID NO:96. In one embodiment, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:97. Inanother embodiment, the peptide sequence comprises an amino acidsequence set forth in SEQ ID NO:98. In other embodiments, the peptidesequence comprises an amino acid sequence set forth in SEQ ID NO:138. Inone embodiment, the peptide sequence comprises an amino acid sequenceset forth in SEQ ID NO:139. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:140. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:141. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:142. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:143. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:144. In oneembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:145. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:146. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:147. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:148. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:149. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:150. In oneembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:151. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:152. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:153. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:154. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:155. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:156. In oneembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:157. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:158. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:159. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:160. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:161. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:162. In oneembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:163. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:164. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:165. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:166. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:167. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:168. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:192. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:193. In oneembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:194. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:195. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:196. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:197. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:198. In other embodiments, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:199. In oneembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:200. In another embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:201. In otherembodiments, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:202. In one embodiment, the peptide sequencecomprises an amino acid sequence set forth in SEQ ID NO:203. In anotherembodiment, the peptide sequence comprises an amino acid sequence setforth in SEQ ID NO:204. In certain embodiments of the various peptidesequences provided herein, the R residue at the N-terminus is deleted.

In yet other embodiments, the peptide sequence consists of an amino acidsequence set forth in SEQ ID NO:1. In another embodiment, the peptidesequence consists of an amino acid sequence set forth in SEQ ID NO:2. Inother embodiments, the peptide sequence consists of an amino acidsequence set forth in SEQ ID NO:3. In one embodiment, the peptidesequence consists of an amino acid sequence set forth in SEQ ID NO:4. Inanother embodiment, the peptide sequence consists of an amino acidsequence set forth in SEQ ID NO:5. In other embodiments, the peptidesequence consists of an amino acid sequence set forth in SEQ ID NO:6. Inone embodiment, the peptide sequence consists of an amino acid sequenceset forth in SEQ ID NO:7. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:8. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:9. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:10. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:11. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:12. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:13. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:14. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:15. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:16. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:17. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:18. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:19. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:20. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:21. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:22. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:23. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:24. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:25. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:26. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:27. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:28. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:29. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:30. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:31. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:32. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:33. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:34. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:35. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:36. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:37. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:38. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:39. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:40. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:41. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:42. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:43. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:44. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:45. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:46. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:47. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:48. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:49. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:50. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:51. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:52. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:53. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:54. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:55. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:56. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:57. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:58. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:59. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:60. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:61. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:62. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:63. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:64. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:65. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:66. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:67. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:68. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:69. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:70. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:71. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:72. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:73. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:74. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:75. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:76. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:77. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:78. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:79. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:80. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:81. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:82. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:83. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:84. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:85. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:86. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:87. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:88. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:89. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:90. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:91. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:92. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:93. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:94. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:95. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:96. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:97. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:98. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:138. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:139. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:140. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:141. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:142. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:143. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:144. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:145. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:146. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:147. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:148. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:149. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:150. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:151. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:152. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:153. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:154. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:155. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:156. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:157. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:158. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:159. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:160. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:161. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:162. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:163. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:164. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:165. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:166. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:167. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:168. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:192. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:193. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:194. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:195. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:196. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:197. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:198. In otherembodiments, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:199. In one embodiment, the peptide sequence consistsof an amino acid sequence set forth in SEQ ID NO:200. In anotherembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:201. In other embodiments, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:202. In oneembodiment, the peptide sequence consists of an amino acid sequence setforth in SEQ ID NO:203. In another embodiment, the peptide sequenceconsists of an amino acid sequence set forth in SEQ ID NO:204. Incertain embodiments of the various peptide sequences provided herein,the R residue at the N-terminus is deleted.

5.4 Particular Modifications to Enhance Peptide Function

It is frequently beneficial, and sometimes imperative, to improve one ofmore physical properties of the treatment modalities disclosed hereinand/or the manner in which they are administered. Improvements ofphysical properties include, for example, modulating immunogenicity;methods of increasing solubility, bioavailability, serum half-life,and/or therapeutic half-life; and/or modulating biological activity.Certain modifications may also be useful to, for example, raise ofantibodies for use in detection assays (e.g., epitope tags) and toprovide for ease of protein purification. Such improvements mustgenerally be imparted without adversely impacting the bioactivity of thetreatment modality and/or increasing its immunogenicity.

Pegylation of is one particular modification contemplated herein, whileother modifications include, but are not limited to, glycosylation (N-and O-linked); polysialylation; albumin fusion molecules comprisingserum albumin (e.g., human serum albumin (HSA), cyno serum albumin, orbovine serum albumin (BSA)); albumin binding through, for example aconjugated fatty acid chain (acylation); and Fc-fusion proteins.

5.4.1 Pegylation

The clinical effectiveness of protein therapeutics is often limited byshort plasma half-life and susceptibility to protease degradation.Studies of various therapeutic proteins (e.g., filgrastim) have shownthat such difficulties may be overcome by, for example, conjugating orlinking the protein to any of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes. This is frequently effected by a linking moietycovalently bound to both the protein and the nonproteinaceous polymer,e.g., a PEG. Such PEG-conjugated biomolecules have been shown to possessclinically useful properties, including better physical and thermalstability, protection against susceptibility to enzymatic degradation,increased solubility, longer in vivo circulating half-life and decreasedclearance, reduced immunogenicity and antigenicity, and reducedtoxicity. In addition to the beneficial effects of pegylation onpharmacokinetic parameters, pegylation itself may enhance activity.

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formulaR(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective group such asan alkyl or an alkanol group, and where n is an integer from 1 to 1000.When R is a protective group, it generally has from 1 to 8 carbons. ThePEG conjugated to the polypeptide sequence can be linear or branched.Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure. A molecular weight of the PEGused in embodiments provided herein is not restricted to any particularrange, and examples are set forth elsewhere herein; by way of example,certain embodiments have molecular weights between 5 kDa and 20 kDa,while other embodiments have molecular weights between 4 kDa and 10 kDa.

In other embodiments, provided herein are compositions of conjugateswherein the PEGs have different n values, and thus the various differentPEGs are present in specific ratios. For example, some compositionscomprise a mixture of conjugates where n=1, 2, 3 and 4. In somecompositions, the percentage of conjugates where n=1 is 18-25%, thepercentage of conjugates where n=2 is 50-66%, the percentage ofconjugates where n=3 is 12-16%, and the percentage of conjugates wheren=4 is up to 5%. Such compositions can be produced by reactionconditions and purification methods know in the art. Cation exchangechromatography may be used to separate conjugates, and a fraction isthen identified which contains the conjugate having, for example, thedesired number of PEGs attached, purified free from unmodified proteinsequences and from conjugates having other numbers of PEGs attached.

Pegylation most frequently occurs at the alpha amino group at theN-terminus of the polypeptide, the epsilon amino group on the side chainof lysine residues, and the imidazole group on the side chain ofhistidine residues. Since most recombinant polypeptides possess a singlealpha and a number of epsilon amino and imidazole groups, numerouspositional isomers can be generated depending on the linker chemistry.

General pegylation strategies known in the art can be applied herein.PEG may be bound to a polypeptide provided herein via a terminalreactive group (a “spacer” or “linker”) which mediates a bond betweenthe free amino or carboxyl groups of one or more of the polypeptidesequences and polyethylene glycol. The PEG having the spacer which maybe bound to the free amino group includes N-hydroxysuccinylimidepolyethylene glycol which may be prepared by activating succinic acidester of polyethylene glycol with N-hydroxysuccinylimide. Anotheractivated polyethylene glycol which may be bound to a free amino groupis 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which maybe prepared by reacting polyethylene glycol monomethyl ether withcyanuric chloride. The activated polyethylene glycol which is bound tothe free carboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences provided hereinto PEG having a spacer may be carried out by various conventionalmethods. For example, the conjugation reaction can be carried out insolution at a pH of from 5 to 10, at temperature from 4° C. to roomtemperature, for 30 minutes to 20 hours, utilizing a molar ratio ofreagent to protein of from 4:1 to 30:1. Reaction conditions may beselected to direct the reaction towards producing predominantly adesired degree of substitution. In general, low temperature, low pH(e.g., pH=5), and short reaction time tend to decrease the number ofPEGs attached, whereas high temperature, neutral to high pH (e.g.,pH≥7), and longer reaction time tend to increase the number of PEGsattached. Various means known in the art may be used to terminate thereaction. In some embodiments, the reaction is terminated by acidifyingthe reaction mixture and freezing at, e.g., −20° C. Pegylation ofvarious molecules is discussed in, for example, U.S. Pat. Nos.5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263.

In some embodiments, also provided herein are uses of PEG mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., XTEN technology; Amunix; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

5.4.2 Glycosylation

As used herein, “glycosylation” is meant to broadly refer to theenzymatic process by which glycans are attached to proteins, lipids orother organic molecules. The use of the term “glycosylation” herein isgenerally intended to mean adding or deleting one or more carbohydratemoieties (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that may or may not be present inthe native sequence. In addition, the phrase includes qualitativechanges in the glycosylation of the native proteins involving a changein the nature and proportions of the various carbohydrate moietiespresent.

Glycosylation can dramatically affect the physical properties (e.g.,solubility) of polypeptides and can also be important in proteinstability, secretion, and subcellular localization. Glycosylatedpolypeptides may also exhibit enhanced stability or may improve one ormore pharmacokinetic properties, such as half-life. In addition,solubility improvements can, for example, enable the generation offormulations more suitable for pharmaceutical administration thanformulations comprising the non-glycosylated polypeptide.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence. The alteration to the polypeptide may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for O-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. A particular embodiment comprisesthe generation and use of N-glycosylation variants.

The polypeptide sequences provided herein may optionally be alteredthrough changes at the nucleic acid level, particularly by mutating thenucleic acid encoding the polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Various cell lines can be used to produce proteins that areglycosylated. One non-limiting example is Dihydrofolate reductase(DHFR)—deficient Chinese Hamster Ovary (CHO) cells, which are a commonlyused host cell for the production of recombinant glycoproteins. Thesecells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

5.4.3 Polysialylation

In certain embodiments, also provided herein is the use ofpolysialylation, the conjugation of polypeptides to the naturallyoccurring, biodegradable α-(2→8) linked polysialic acid (“PSA”) in orderto improve the polypeptides' stability and in vivo pharmacokinetics.

Albumin Fusion:

Additional suitable components and molecules for conjugation includealbumins such as human serum albumin (HSA), cyno serum albumin, andbovine serum albumin (BSA).

In some embodiments, albumin is conjugated to a drug molecule (e.g., apolypeptide described herein) at the carboxyl terminus, the aminoterminus, both the carboxyl and amino termini, and internally (see,e.g., U.S. Pat. Nos. 5,876,969 and 7,056,701).

In the HSA—drug molecule conjugates embodiments provided herein, variousforms of albumin may be used, such as albumin secretion pre-sequencesand variants thereof, fragments and variants thereof, and HSA variants.Such forms generally possess one or more desired albumin activities. Inadditional embodiments, fusion proteins are provided herein comprising apolypeptide drug molecule fused directly or indirectly to albumin, analbumin fragment, an albumin variant, etc., wherein the fusion proteinhas a higher plasma stability than the unfused drug molecule and/or thefusion protein retains the therapeutic activity of the unfused drugmolecule. In some embodiments, the indirect fusion is effected by alinker, such as a peptide linker or modified version thereof.

As alluded to above, fusion of albumin to one or more polypeptidesprovided herein can, for example, be achieved by genetic manipulation,such that the nucleic acid coding for HSA, or a fragment thereof, isjoined to the nucleic acid coding for the one or more polypeptidesequences.

5.4.4 Alternative Albumin Binding Strategies

Several albumin—binding strategies have been developed as alternativesto direct fusion and may be used with the agents described herein. Byway of example, in certain embodiments, provided herein is albuminbinding through a conjugated fatty acid chain (acylation) and fusionproteins which comprise an albumin binding domain (ABD) polypeptidesequence and the sequence of one or more of the polypeptides describedherein.

Fusion of albumin to a peptide sequence can, for example, be achieved bygenetic manipulation, such that the DNA coding for HSA (human serumalbumin), or a fragment thereof, is joined to the DNA coding for apeptide sequence. Thereafter, a suitable host can be transformed ortransfected with the fused nucleotide sequence in the form of, forexample, a suitable plasmid, so as to express a fusion polypeptide. Theexpression may be effected in vitro from, for example, prokaryotic oreukaryotic cells, or in vivo from, for example, a transgenic organism.In some embodiments, the expression of the fusion protein is performedin mammalian cell lines, for example, CHO cell lines.

Further means for genetically fusing target proteins or peptides toalbumin include a technology known as Albufuse® (Novozymes BiopharmaA/S; Denmark), and the conjugated therapeutic peptide sequencesfrequently become much more effective with better uptake in the body.The technology has been utilized commercially to produce Albuferon®(Human Genome Sciences), a combination of albumin and interferon α-2Bused to treat hepatitis C infection.

Another embodiment entails the use of one or more human domainantibodies (dAb). dAbs are the smallest functional binding units ofhuman antibodies (IgGs) and have favorable stability and solubilitycharacteristics. The technology entails a dAb(s) conjugated to HSA(thereby forming a “AlbudAb”; see, e.g., EP1517921B, WO2005/118642 andWO2006/051288) and a molecule of interest (e.g., a peptide sequenceprovided herein). AlbudAbs are often smaller and easier to manufacturein microbial expression systems, such as bacteria or yeast, than currenttechnologies used for extending the serum half-life of peptides. As HSAhas a half-life of about three weeks, the resulting conjugated moleculeimproves the half-life. Use of the dAb technology may also enhance theefficacy of the molecule of interest.

5.4.5 Conjugation with Other Molecules

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; tetanus toxoid; Diphtheria toxoid; polyaminoacids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides ofrotaviruses; influenza virus hemagglutinin, influenza virusnucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B viruscore protein and surface antigen; or any combination of the foregoing.

Thus, in certain embodiments, conjugation of one or more additionalcomponents or molecules at the N- and/or C-terminus of a polypeptidesequence, such as another polypeptide (e.g., a polypeptide having anamino acid sequence heterologous to the subject polypeptide), or acarrier molecule is also contemplated. Thus, an exemplary polypeptidesequence can be provided as a conjugate with another component ormolecule.

A polypeptide may also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, or cellulose beads; polymeric amino acids such aspolyglutamic acid, or polylysine; amino acid copolymers; inactivatedvirus particles; inactivated bacterial toxins such as toxoid fromdiphtheria, tetanus, cholera, or leukotoxin molecules; inactivatedbacteria; and dendritic cells. Such conjugated forms, if desired, can beused to produce antibodies against a polypeptide provided herein.

5.4.6 Fc-Fusion Molecules

In certain embodiments, the amino- or carboxyl-terminus of a polypeptidesequence provided herein is fused with an immunoglobulin Fc region toform a fusion conjugate (or fusion molecule). In a specific embodiment,the immunoglobulin Fc region is a human Fc region. Fusion conjugateshave been shown to increase the systemic half-life ofbiopharmaceuticals, and thus the biopharmaceutical product may requireless frequent administration. In certain embodiments, the half-life isincreased as compared to the same polypeptide that is not fused to animmunoglobulin Fc region.

Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells thatline the blood vessels, and, upon binding, the Fc fusion molecule isprotected from degradation and re-released into the circulation, keepingthe molecule in circulation longer. This Fc binding is believed to bethe mechanism by which endogenous IgG retains its long plasma half-life.More recent Fc-fusion technology links a single copy of abiopharmaceutical to the Fc region of an antibody to optimize thepharmacokinetic and pharmacodynamic properties of the biopharmaceuticalas compared to traditional Fc-fusion conjugates.

In some embodiments, provided herein is a fusion of M70 to a humanantibody Fc fragment. In some embodiments, provided herein is a fusionof M69 to a human antibody Fc fragment. Such fusions can be useful inthe treatment of bile acid related disorders and other metabolicdisorders provided herein. In some embodiments, the Fc-fusion of M70 hasa longer half-life. In specific embodiments, the longer half-life of theFc-fusion of M70 is as compared to M70 that is not an Fc-fusion. In someembodiments, the Fc-fusion of M69 has a longer half-life. In specificembodiments, the longer half life of the Fc-fusion of M69 is as comparedto M69 that is not an Fc-fusion. Such a long half-life makes thesefusions suitable for once weekly, or less frequent dosing.

In some embodiments, the Fc-fusion comprises a linker. Exemplaryflexible linkers include glycine polymers (G_()n), glycine-serinepolymers, glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers. In certain embodiments, the linker is (G)₄S. In someembodiments, the linker is ((G)₄S)_(n), where n is an integer of atleast one. In some embodiments, the linker is ((G)₄S)₂. Glycine andglycine-serine polymers are relatively unstructured, and therefore mayserve as a neutral tether between components. In some embodiments, theglycine-serine polymer is (GS)_(n), where n is an integer of at leastone. In some embodiments, the glycine-serine polymer is GSGGS_(n) (SEQID NO:129), where n is an integer of at least one. In some embodiments,the glycine-serine polymer is GGGS_(n) (SEQ ID NO:130), where n is aninteger of at least one. In certain embodiments, the linker comprises anadditional G residue at the N′ terminus of SEQ ID NO:130. In oneembodiment, the linker is GGSG (SEQ ID NO:131). In one embodiment, thelinker is GGSGG (SEQ ID NO:132). In one embodiment, the linker is GSGSG(SEQ ID NO:133). In one embodiment, the linker is GSGGG (SEQ ID NO:134).In one embodiment, the linker is GGGSG (SEQ ID NO:189). In oneembodiment, the linker is GSSSG (SEQ ID NO:135).

5.4.7 Purification

Additional suitable components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH ˜4, and then eluted with a linear (0 M to 0.5 M) NaClgradient buffered at a pH from 3 to 5.5, such as at pH ˜4.5. The contentof the fractions obtained by cation exchange chromatography may beidentified by molecular weight using conventional methods, for example,mass spectroscopy, SDS-PAGE, or other known methods for separatingmolecular entities by molecular weight. A fraction is then identifiedwhich contains the conjugate having the desired number of PEGs attached,purified free from unmodified protein sequences and from conjugateshaving other numbers of PEGs attached.

5.4.8 Other Modifications

In certain embodiments, also provided herein is the use of othermodifications, currently known or developed in the future, to improveone or more properties. Examples include hesylation, various aspects ofwhich are described in, for example, U.S. Patent Appln. Nos.2007/0134197 and 2006/0258607, and fusion molecules comprising SUMO as afusion tag (LifeSensors, Inc.; Malvern, Pa.).

In still other embodiments, a peptide sequence provided herein is linkedto a chemical agent (e.g., an immunotoxin or chemotherapeutic agent),including, but are not limited to, a cytotoxic agent, including taxol,cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, andanalogs or homologs thereof. Other chemical agents include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine); alkylating agents (e.g.,mechlorethamine, carmustine and lomustine, cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cisplatin);antibiotics (e.g., bleomycin); and anti-mitotic agents (e.g.,vincristine and vinblastine). Cytotoxins can be conjugated to a peptideprovided herein using linker technology known in the art and describedherein.

Further suitable components and molecules for conjugation include thosesuitable for detection in an assay. Particular non-limiting examplesinclude detectable labels, such as a radioisotope (e.g., ¹²⁵I; ³⁵S, ³²P;³³P), an enzyme which generates a detectable product (e.g., luciferase,β-galactosidase, horse radish peroxidase and alkaline phosphatase), afluorescent protein, a chromogenic protein, dye (e.g., fluoresceinisothiocyanate); fluorescence emitting metals (e.g. ¹⁵²Eu);chemiluminescent compounds (e.g., luminol and acridinium salts);bioluminescent compounds (e.g., luciferin); and fluorescent proteins.Indirect labels include labeled or detectable antibodies that bind to apeptide sequence, where the antibody may be detected.

In certain embodiments, a peptide sequence provided herein is conjugatedto a radioactive isotope to generate a cytotoxic radiopharmaceutical(radioimmunoconjugates) useful as a diagnostic or therapeutic agent.Examples of such radioactive isotopes include, but are not limited to,iodine¹³¹, indium¹¹¹, yttrium⁹⁰ and lutetium¹⁷⁷. Methods for preparingradioimmunoconjugates are known to the skilled artisan. Examples ofradioimmunoconjugates that are commercially available includeibritumomab, tiuxetan, and tositumomab.

5.4.9 Linkers

Linkers and their use have been described above. Any of the foregoingcomponents and molecules used to modify the polypeptide sequencesprovided herein may optionally be conjugated via a linker. Suitablelinkers include “flexible linkers” which are generally of sufficientlength to permit some movement between the modified polypeptidesequences and the linked components and molecules. The linker moleculesare generally about 6-50 atoms long. The linker molecules may also be,for example, aryl acetylene, ethylene glycol oligomers containing 2-10monomer units, diamines, diacids, amino acids, or combinations thereof.Suitable linkers can be readily selected and can be of any suitablelength, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30, 30-50 or more than 50 amino acids.

Exemplary flexible linkers include glycine polymers (G_()n),glycine-serine polymers (for example, (GS)_(n), GSGGS_(n) (SEQ IDNO:129) and GGGS_(n) (SEQ ID NO:130), where n is an integer of at leastone), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers. Glycine and glycine-serine polymers are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Exemplary flexible linkers include, but are not limited toGGSG (SEQ ID NO:131), GGSGG (SEQ ID NO:132), GSGSG (SEQ ID NO:133),GSGGG (SEQ ID NO:134), GGGSG (SEQ ID NO:189), and GSSSG (SEQ ID NO:135).In certain embodiments, the linker is (G)₄S. In some embodiments, thelinker is ((G)₄S)_(n)), where n is an integer of at least one. In someembodiments, the linker is ((G)₄S)₂). In some embodiments, theglycine-serine polymer is (GS)_(n), where n is an integer of at leastone. In some embodiments, the glycine-serine polymer is GSGGS_(n) (SEQID NO:129), where n is an integer of at least one. In some embodiments,the glycine-serine polymer is GGGS_(n) (SEQ ID NO:130), where n is aninteger of at least one. In certain embodiments, the linker comprises anadditional G residue at the N′ terminus of SEQ ID NO:130. In oneembodiment, the linker is GGSG (SEQ ID NO:131). In one embodiment, thelinker is GGSGG (SEQ ID NO:132). In one embodiment, the linker is GSGSG(SEQ ID NO:133). In one embodiment, the linker is GSGGG (SEQ ID NO:134).In one embodiment, the linker is GGGSG (SEQ ID NO:189). In oneembodiment, the linker is GSSSG (SEQ ID NO:135).

Peptide sequences provided herein, including the FGF19 and FGF21variants and subsequences and the FGF19/FGF21 fusions and chimeraslisted in Table 1 and Sequence Listing, as well as subsequences,sequence variants and modified forms of the sequences listed in Table 1and Sequence Listing have one or more activities as set forth herein.One example of an activity is modulating bile acid homeostasis. Anotherexample of an activity is reduced stimulation or formation of HCC, forexample, as compared to FGF19. An additional example of an activity islower or reduced lipid (e.g., triglyceride, cholesterol, non-HDL) or HDLincreasing activity, for example, as compared to FGF21. A furtherexample of an activity is a lower or reduced lean muscle mass reducingactivity, for example, as compared to FGF21. Yet another example of anactivity is binding to FGFR4, or activating FGFR4, for example, peptidesequences that bind to FGFR4 with an affinity comparable to or greaterthan FGF19 binding affinity for FGFR4; and peptide sequences thatactivate FGFR4 to an extent or amount comparable to or greater thanFGF19 activates FGFR4. Still further examples of activities includetreating a bile acid-related or associated disorder. Activities such as,for example, modulation of bile acid homeostasis, glucose loweringactivity, analysis of a bile acid-related or associated disorder, HCCformation or tumorigenesis, lipid increasing activity, or lean massreducing activity can be ascertained in an animal, such as a db/dbmouse. Measurement of binding to FGFR4 or activation of FGFR4 can beascertained by assays disclosed herein or known to the skilled artisan.

Various methodologies can be used in the screening and diagnosis of HCCand are well known to the skilled artisan. Indicators for HCC includedetection of a tumor maker such as elevated alpha-fetoprotein (AFP) ordes-gamma carboxyprothrombin (DCP) levels. A number of differentscanning and imaging techniques are also helpful, including ultrasound,CT scans and MRI. In certain embodiments, evaluation of whether apeptide (e.g., a candidate peptide) exhibits evidence of inducing HCCmay be determined in vivo by, for example, quantifying HCC noduleformation in an animal model, such as db/db mice, administered apeptide, compared to HCC nodule formation by wild type FGF19.Macroscopically, liver cancer may be nodular, where the tumor nodules(which are round-to-oval, grey or green, well circumscribed but notencapsulated) appear as either one large mass or multiple smallermasses. Alternatively, HCC may be present as an infiltrative tumor whichis diffuse and poorly circumscribed and frequently infiltrates theportal veins. Pathological assessment of hepatic tissue samples isgenerally performed after the results of one or more of theaforementioned techniques indicate the likely presence of HCC. Thus,methods provided herein may further include assessing a hepatic tissuesample from an in vivo animal model (e.g., a db/db mouse) useful in HCCstudies in order to determine whether a peptide sequence exhibitsevidence of inducing HCC. By microscopic assessment, a pathologist candetermine whether one of the four general architectural and cytologicaltypes (patterns) of HCC are present (i.e., fibrolamellar,pseudoglandular (adenoid), pleomorphic (giant cell) and clear cell).

More particularly, peptide sequences provided herein, including theFGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusionsand chimeras listed in Table 1 and Sequence Listing, as well assubsequences, variants and modified forms of the sequences listed inTable 1 and Sequence Listing include those with the followingactivities: peptide sequences modulating bile acid homeostasis ortreating a bile acid-related or associated disorder while having reducedHCC formation compared to FGF19, or a FGF 19 variant sequence having anyof GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ IDNO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ IDNO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ IDNO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ IDNO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted forthe WGDPI (SEQ ID NO:170) sequence at amino acids 16-20 of FGF19;peptide sequences having greater bile acid modulating activity comparedto FGF19, or FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19; peptide sequences havingless lipid increasing activity (e.g., less triglyceride, cholesterol,non-HDL) or more HDL increasing activity compared to FGF19, or a FGF 19variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19; and peptide sequences having less lean massreducing activity as compared to FGF21.

More particularly, peptide sequences provided herein, including theFGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusionsand chimeras listed in Table 1 and Sequence Listing, as well assubsequences, variants and modified forms of the sequences listed inTable 1 and the Sequence Listing include those with the followingactivities: peptide sequences that modulate bile acid homeostasis;peptide sequences that treat a bile acid-related or associated disorder,peptide sequences that bind to FGFR4, or activate FGFR4, such as peptidesequences that bind to FGFR4 with an affinity comparable to or greaterthan FGF19 binding affinity for FGFR4; peptide sequences that activateFGFR4 to an extent or amount comparable to or greater than FGF19activates FGFR4; peptide sequences that down-regulate or reducealdo-keto reductase gene expression, for example, compared to FGF19; andpeptide sequences that up-regulate or increase solute carrier family 1,member 2 (Slc1a2) gene expression as compared to FGF21.

As disclosed herein, variants include various N-terminal modificationsand/or truncations of FGF19, including variants in which there has beena substitution of one or several N-terminal FGF19 amino acids with aminoacids from FGF21. Such variants include variants having glucose loweringactivity, as well as a favorable lipid profile and are not measurably ordetectably tumorigenic.

5.5 Dosing and Administration

Peptide sequences provided herein including subsequences, sequencevariants and modified forms of the exemplified peptide sequences (e.g.,sequences listed in the Sequence Listing or Table 1), may be formulatedin a unit dose or unit dosage form. In a particular embodiment, apeptide sequence is in an amount effective to treat a subject in need oftreatment, e.g., due to abnormal or aberrant bile acid homeostasis, suchas metabolic syndrome; a lipid- or glucose-related disorder; cholesterolor triglyceride metabolism; type 2 diabetes; cholestasis, including, forexample diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension. Exemplary unit doses range from about 25-250, 250-500,500-1000, 1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000 ng; fromabout 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,25,000-50,000 μg; and from about 25-250, 250-500, 500-1000, 1000-2500 or2500-5000, 5000-25,000, 25,000-50,000 mg.

Peptide sequences provided herein including subsequences, sequencevariants and modified forms of the exemplified peptide sequences (e.g.,sequences listed in the Sequence Listing or Table 1) can be administeredto provide the intended effect as a single dose or multiple dosages, forexample, in an effective or sufficient amount. Exemplary doses rangefrom about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000,5000-25,000, 25,000-50,000 pg/kg; from about 50-500, 500-5000,5000-25,000 or 25,000-50,000 ng/kg; and from about 25-250, 250-500,500-1000, 1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000 μg/kg.Single or multiple doses can be administered, for example, multipletimes per day, on consecutive days, alternating days, weekly orintermittently (e.g., twice per week, once every 1, 2, 3, 4, 5, 6, 7 or8 weeks, or once every 2, 3, 4, 5 or 6 months).

Peptide sequences provided herein including subsequences, variants andmodified forms of the exemplified peptide sequences (e.g., sequenceslisted in the Sequence Listing or Table 1) can be administered andmethods may be practiced via systemic, regional or local administration,by any route. For example, a peptide sequence can be administeredparenterally (e.g., subcutaneously, intravenously, intramuscularly, orintraperitoneally), orally (e.g., ingestion, buccal, or sublingual),inhalation, intradermally, intracavity, intracranially, transdermally(topical), transmucosally or rectally. Peptide sequences provided hereinincluding subsequences, variants and modified forms of the exemplifiedpeptide sequences (e.g., sequences listed in the Sequence Listing orTable 1) and methods provided herein including pharmaceuticalcompositions can be administered via a (micro)encapsulated deliverysystem or packaged into an implant for administration.

A particular non-limiting example of parenteral (e.g., subcutaneous)administration entails the use of Intarcia's subcutaneous deliverysystem (Intarcia Therapeutics, Inc.; Hayward, Calif.). The systemcomprises a miniature osmotic pump that delivers a consistent amount ofa therapeutic agent over a desired period of time. In addition tomaintaining drug levels within an appropriate therapeutic range, thesystem can be used with formulations that maintain the stability ofproteinaceous therapeutic agents at human body temperature for extendedperiods of time.

Another non-limiting example of parenteral administration entails theuse of DUROS®-type implantable osmotic pumps (from, e.g., DURECT Corp.).The DUROS® system can be used for therapies requiring systemic orsite-specific administration of a drug. To deliver drugs systemically,the DUROS® system is placed just under the skin, for example in theupper arm, in an outpatient procedure that is completed in just a fewminutes using local anesthetic. To deliver a drug to a specific site,miniaturized catheter technology can be used. The catheter can beattached to the DUROS® system to direct the flow of a drug to the targetorgan, tissue or synthetic medical structure, such as a graft.Site-specific delivery enables a therapeutic concentration of a drug tobe administered to the desired target without exposing the entire bodyto a similar concentration. The precision, size and performance of theDUROS® system will allow for continuous site-specific delivery to avariety of precise locations within the body.

Yet another non-limiting example of parenteral administration entailsthe use of an on-body delivery system (e.g., the Neulasta® Delivery Kitby Amgen). This on-body delivery system includes an on-body injector,which is a small, lightweight, injection system applied on the same dayas a doctor visit (such as the day of chemotherapy). It is designed todeliver a dose of the therapeutic agent the next day, or in the nearfuture of the doctor visit, so that the patient does not need to returnto the doctor's office to receive the injection.

5.6 Methods of Preventing, Treating and Managing Diseases and Disorders

In one embodiment, provided herein is a method of preventing a diseaseor disorder in a subject having, or at risk of having, a disease ordisorder preventable by a peptide sequence provided herein, comprisingadministering a pharmaceutical composition comprising a peptide providedherein to a subject in an amount effective for preventing the disease ordisorder. In another embodiment, provided herein is a method of treatinga disease or disorder in a subject having, or at risk of having, adisease or disorder treatable by a peptide sequence provided herein,comprising administering a pharmaceutical composition comprising apeptide provided herein to a subject in an amount effective for treatingthe disease or disorder. In yet another embodiment, provided herein is amethod of managing a disease or disorder in a subject having, or at riskof having, a disease or disorder manageable by a peptide sequenceprovided herein, comprising administering a pharmaceutical compositioncomprising a peptide provided herein to a subject in an amount effectivefor managing the disease or disorder. In one embodiment, the disease ordisorder is a bile acid-related disease or associated disorder. Inanother embodiment, the disease or disorder is a metabolic disease ordisorder. In other embodiments, the disease or disorder is a cancer ortumor.

Administration of various FGF19 and/FGF21 variants and fusion peptidesequences to mice successfully modulated bile acid homeostasis andhyperglycemia (data not shown). Furthermore, in contrast to FGF19,certain peptide sequences did not stimulate or induce HCC formation ortumorigenesis in mice (data not shown). Thus, administration of peptidesprovided herein, including subsequences, variants and modified forms ofthe exemplified peptide sequences (including the FGF19 and FGF21variants and subsequences listed in Table 1 and the Sequence Listing,and the FGF19/FGF21 fusions and chimeras listed in Table 1 and theSequence Listing), into an animal, either by direct or indirect in vivoor by ex vivo methods (e.g., administering the variant or fusionpeptide, a nucleic acid encoding the variant or fusion peptide, or atransformed cell or gene therapy vector expressing the variant or fusionpeptide), can be used to treat various disorders, such as bile-acidrelated or associated disorders, and metabolic disorders, such asdisorders related to high sugar levels, hyperglycemic conditions,insulin resistance, hyperinsulinemia, glucose intolerance, metabolicsyndrome, or related disorders, as set forth herein,

5.6.1 Methods of Preventing, Treating and Managing Bile Acid-Related orAssociated Disorders

As used herein, the phrases “bile acid-related disorder,” “bileacid-related or associated disorder,” and the like, when used inreference to a condition of a subject, means a disruption of bile acidhomeostasis, which may manifest itself as, for example, an acute,transient or chronic abnormal level of a bile acid or one or more bileacids. The condition can be caused by inhibition, reduction or a delayin bile acid synthesis, metabolism or absorption such that the subjectexhibits a bile acid level not typically found in normal subjects.

Also provided herein are in vitro, ex vivo and in vivo (e.g., on or in asubject) methods and uses. Such methods and uses can be practiced withany of the peptide sequences set forth herein. In various embodiments,the methods include administering a peptide sequence, such as a FGF19 orFGF21 variant, fusion or chimera disclosed herein (e.g., in the SequenceListing or Table 1), or a subsequence, a variant or modified form of aFGF19 or FGF21 variant, fusion or chimera disclosed herein (e.g., theSequence Listing or Table 1), to a subject in an amount effective fortreating a bile acid-related or associated disorder.

In certain embodiments, the peptide is administered in combination withan additional therapeutic agent(s) and/or treatment modalities (e.g., anagent useful in the treatment and/or prevention of PBC). The additionaltherapeutic agent(s) can be administered before, with, or followingadministration of the peptides described herein.

Also provided here are methods of preventing (e.g., in subjectspredisposed to having a particular disorder(s)), delaying, slowing orinhibiting progression of, the onset of, or treating (e.g.,ameliorating) a bile acid-related or associated disorder relative to anappropriate matched subject of comparable age, gender, race, etc.).Thus, in various embodiments, a method provided herein for, for example,modulating bile acid homeostasis or treating a bile acid-related orassociated disorder includes contacting or administering one or morepeptides provided herein (e.g., a variant or fusion of FGF19 and/orFGF21 as set forth in the Sequence Listing or Table 1) in an amounteffective to modulate bile acid homeostasis or treat a bile acid-relatedor associated disorder. In certain embodiments the method furthercomprises contacting or administering at least one additionaltherapeutic agent or treatment modality that is useful in the treatmentor prevention of a bile acid-related or associated disorder (e.g., PBC).

The term “subject” refers to an animal. Typically, the animal is amammal that would benefit from treatment with a peptide sequenceprovided herein. Particular examples include primates (e.g., humans),dogs, cats, horses, cows, pigs, and sheep.

Subjects include those having a disorder, e.g., a bile acid-related orassociated disorder, such as cholestasis, including, for examplediseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), short bowel syndrome, disorders impairingabsorption of bile acids not otherwise characterized (idiopathic))leading to diarrhea (e.g., BAD) and GI symptoms, and GI, liver, and/orbiliary cancers (e.g., colon cancer and hepatocellular cancer); and/orbile acid synthesis abnormalities, such as those contributing to NASH,cirrhosis and portal hypertension; or subjects that do not have adisorder but may be at risk of developing the disorder.

Non-limiting exemplary bile acid-related or associated disorderspreventable, treatable or manageable according to the methods and usesprovided herein include: cholestasis, including, for example diseases ofintrahepatic cholestasis (e.g., primary biliary cirrhosis (PBC), primaryfamilial intrahepatic cholestasis (PFIC) (e.g., progressive PFIC),primary sclerosing choangitis (PSC), pregnancy intrahepatic cholestasis(PIC), neonatal cholestasis, and drug-induced cholestasis (e.g.,estrogen)), and diseases of extrahepatic cholestasis (e.g., bile cutcompression from tumor, bile duct blockade by gall stones); bile acidmalabsorption and other disorders involving the distal small intestine,including ileal resection, inflammatory bowel diseases (e.g., Crohn'sdisease and ulcerative colitis), short bowel syndrome, disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., bile acid diarrhea (BAD)) andGI symptoms, and GI, liver, and/or biliary cancers (e.g., colon cancerand hepatocellular cancer); and/or bile acid synthesis abnormalities,such as those contributing to non-alcoholic steatohepatitis (NASH),cirrhosis and portal hypertension; e.g., in mammals, such as humans.Additional bile acid-related or associated disorders include metabolicsyndrome; a lipid or glucose disorder; cholesterol or triglyceridemetabolism; type 2 diabetes. In one particular embodiment, the bileacid-related or associated disorder is bile acid malabsorption. Inanother particular embodiment, the bile acid-related or associateddisorder is diarrhea. In a still further particular embodiment, the bileacid-related or associated disorder is cholestasis (e.g., intrahepaticor extrahepatic cholestasis). In another further particular embodiment,the bile acid-related or associated disorder is primary biliarycirrhosis (PBC). In other particular embodiments, the bile acid-relatedor associated disorder is primary sclerosing cholangitis. In anotherembodiment, the bile acid-related or associated disorder is PFIC (e.g.,progressive PFIC). In another embodiment, the bile acid-related orassociated disorder is NASH. In another embodiment, the bileacid-related or associated disorder is a hyperglycemic condition. In aspecific embodiment, the bile acid-related or associated disorder istype 2 diabetes.

In some embodiments, the pharmaceutical composition further comprises atleast one additional agent effective in modulating bile acid homeostasisor treating a bile acid-related or associated disorder, wherein theadditional agent is: a glucocorticoid; CDCA; UDCA; insulin, an insulinsecretagogues, an insulin mimetic, a sulfonylurea and a meglitinide; abiguanide; an alpha-glucosidase inhibitors; a DPP-IV inhibitor, GLP-1, aGLP-1 agonists and a GLP-1 analog; a DPP-IV-resistant analogue; a PPARgamma agonist, a dual-acting PPAR agonist, a pan-acting PPAR agonist; aPTP1B inhibitor; an SGLT inhibitor; an RXR agonist; a glycogen synthasekinase-3 inhibitor; an immune modulator; a beta-3 adrenergic receptoragonist; an 11beta-HSD1 inhibitor; amylin and an amylin analogue; a bileacid sequestrant; or an SGLT-2 inhibitor. In certain embodiments, the atleast one additional agent effective in modulating PBC is UDCA, an FXRagonist, OCA, an ASBT inhibitor, an autoimmune agent, an anti-IL-12agent, an anti-CD80 agent, an anti-CD20 agent, a CXCL10 neutralizingantibody, a ligand for CXCR3, a fibrate, fish oil, colchicine,methotrexate, azathioprine, cyclosporine, or an anti-retroviral therapy.In particular embodiments, the at least one additional agent effectivein modulating PBC is UDCA, OCA, an ASBT inhibitor, an anti-IL-12 agent,an anti-CD20 agent, or a fibrate.

Additional bile acid-related or associated disorders that may be treatedor prevented with the peptide sequences provided herein includemetabolic syndrome, a lipid or glucose disorder, cholesterol ortriglyceride metabolism, diabetes (e.g., type 2 diabetes), otherhyperglycemic-related disorders, including kidney damage (e.g., tubuledamage or nephropathy), liver degeneration, eye damage (e.g., diabeticretinopathy or cataracts), and diabetic foot disorders, anddyslipidemias and their sequelae such as, for example, atherosclerosis,coronary artery disease, cerebrovascular disorders and the like.

Other conditions which may be associated with metabolic syndrome, suchas obesity and elevated body mass (including the co-morbid conditionsthereof such as, but not limited to, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovariansyndrome (PCOS)), and also include thromboses, hypercoagulable andprothrombotic states (arterial and venous), hypertension (includingportal hypertension (defined as a hepatic venous pressure gradient(HVPG) greater than 5 mm Hg), cardiovascular disease, stroke and heartfailure; Disorders or conditions in which inflammatory reactions areinvolved, including atherosclerosis, chronic inflammatory bowel diseases(e.g., Crohn's disease and ulcerative colitis), asthma, lupuserythematosus, arthritis, or other inflammatory rheumatic disorders;Disorders of cell cycle or cell differentiation processes such asadipose cell tumors, lipomatous carcinomas including, for example,liposarcomas, solid tumors, and neoplasms; Neurodegenerative diseasesand/or demyelinating disorders of the central and peripheral nervoussystems and/or neurological diseases involving neuroinflammatoryprocesses and/or other peripheral neuropathies, including Alzheimer'sdisease, multiple sclerosis, Parkinson's disease, progressive multifocalleukoencephalopathy and Guillian-Barre syndrome; Skin and dermatologicaldisorders and/or disorders of wound healing processes, includingerythemato-squamous dermatoses; and Other Disorders such as syndrome X,osteoarthritis, and acute respiratory distress syndrome.

Treatment of a bile acid-related or associated disorder (e.g., NASH) mayhave the benefit of alleviating or abolishing a disorder secondarythereto. By way of example, a subject suffering from NASH may also havedepression or anxiety due to NASH; thus, treating the subject's NASH mayalso indirectly treat the depression or anxiety. The use of thetherapies disclosed herein to target such secondary disorders is alsocontemplated in certain embodiments.

In particular embodiments, the subject has or is at risk of having PBC.In other particular embodiments, the subject has or is at risk of havingNASH.

Subjects at risk of developing a bile acid-related or associateddisorder (such as the disorders described above) include, for example,those who may have a family history or genetic predisposition towardsuch disorder, as well those whose diet may contribute to development ofsuch disorders.

As disclosed herein, treatment methods include contacting oradministering a peptide as set forth herein (e.g., a variant or fusionof FGF19 and/or FGF21 as set forth in the Sequence Listing or Table 1)in an amount effective to achieve a desired outcome or result in asubject. A treatment that results in a desired outcome or resultincludes decreasing, reducing or preventing the severity or frequency ofone or more symptoms of the condition in the subject, e.g., animprovement in the subject's condition or a “beneficial effect” or“therapeutic effect.” Therefore, treatment can decrease or reduce orprevent the severity or frequency of one or more symptoms of thedisorder, stabilize or inhibit progression or worsening of the disorder,and in some instances, reverse the disorder, transiently (e.g., for 1-6,6-12, or 12-24 hours), for medium term (e.g., 1-6, 6-12, 12-24 or 24-48days) or long term (e.g., for 1-6, 6-12, 12-24, 24-48 weeks, or greaterthan 24-48 weeks). Thus, in the case of a bile acid-related orassociated disorder, treatment can lower or reduce one or more symptomsor effects of the bile acid-related or associated disorders describedabove.

In certain embodiments, the various methods provided herein furtherinclude contacting or administering one or more additional agents ortherapeutic modalities useful in the treatment or prevention of a bileacid-related or associated disorder, such as those agents or therapeuticmodalities described herein, in an amount effective to achieve a desiredoutcome or result in a subject.

An “effective amount” or a “sufficient amount” for use and/or fortreating a subject refers to an amount that provides, in single ormultiple doses, alone, or in combination with one or more other agents,treatments, protocols, or therapeutic regimens, a detectable response ofany duration of time (transient, medium or long term), a desired outcomein or an objective or subjective benefit to a subject of any measurableor detectable degree or for any duration of time (e.g., for hours, days,months, years, in remission or cured). Such amounts typically areeffective to ameliorate a disorder, or one, multiple or all adversesymptoms, consequences or complications of the disorder, to a measurableextent, although reducing or inhibiting a progression or worsening ofthe disorder, is considered a satisfactory outcome.

As used herein, the term “ameliorate” means an improvement in thesubject's disorder, a reduction in the severity of the disorder, or aninhibition of progression or worsening of the disorder (e.g.,stabilizing the disorder). In the case of a bile acid-related orassociated disorder such as those described above, including cholestasis(e.g., PBC), disorders impairing absorption of bile acids leading todiarrhea (e.g., BAD) and bile acid synthesis abnormalities (e.g., NASH),an improvement can be a lowering or a reduction in one or more symptomsor effects of the disorder.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the disorder or disease. Thus, asatisfactory endpoint is achieved when there is a transient, medium orlong term, incremental improvement in a subject's condition, or apartial reduction in the occurrence, frequency, severity, progression,or duration, or inhibition or reversal, of one or more associatedadverse symptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the condition, disorder or disease), of the disorder ordisease, over a duration of time (hours, days, weeks, months, etc.).

Thus, in the case of a disorder treatable by a peptide sequence providedherein, either alone or in combination with an additional agent, theamount of the peptide (and optionally the additional agent) sufficientto ameliorate a disorder will depend on the type, severity and extent,or duration of the disorder, the therapeutic effect or outcome desired,and can be readily ascertained by the skilled artisan. Appropriateamounts will also depend upon the individual subject (e.g., thebioavailability within the subject, gender, age, etc.). For example, atransient, or partial, restoration of normal bile acid homeostasis in asubject can reduce the dosage amount or frequency of the peptides andagents described herein in order to treat the bile acid-related orassociated disorders described previously even though complete freedomfrom treatment has not resulted. An effective amount can be ascertained,for example, by measuring one or more relevant physiological effects.

Methods and uses provided herein for treating a subject are applicablefor prophylaxis to prevent or reduce the likelihood of a disorder in asubject, such as a bile acid-related or associated disorder.Accordingly, methods and uses provided herein for treating a subjecthaving, or at risk of developing, a bile acid-related or associateddisorder can be practiced prior to, substantially contemporaneouslywith, or following administration or application of another agent usefulfor the treatment or prevention of a bile acid-related or associateddisorder, and/or can be supplemented with other forms of therapy.Supplementary therapies include other glucose lowering treatments, suchas insulin, an insulin sensitivity enhancer and other drug treatments, achange in diet (low sugar, fats, etc.), weight loss surgery- (reducingstomach volume by gastric bypass, gastrectomy), gastric banding, gastricballoon, gastric sleeve, etc. For example, a method or use providedherein for treating a hyperglycemic or insulin resistance disorder canbe used in combination with drugs or other pharmaceutical compositionsthat lower glucose or increase insulin sensitivity in a subject.

In one embodiment, a method or use includes contacting or administeringto a subject one or more variant or fusion FGF19 and/or FGF21 peptidesequences in an amount effective for preventing a bile-acid related orassociated disorder. In one embodiment, a method or use includescontacting or administering to a subject one or more variant or fusionFGF19 and/or FGF21 peptide sequences in an amount effective for treatinga bile-acid related or associated disorder. In one embodiment, a methodor use includes contacting or administering to a subject one or morevariant or fusion FGF19 and/or FGF21 peptide sequences in an amounteffective for managing a bile-acid related or associated disorder.

5.6.1.1 PBC and Therapy with Agents Effective in the Treatment orPrevention Thereof

Primary biliary cirrhosis (PBC), the most common cholestatic liverdisease, is a progressive hepatic disease that primarily results fromautoimmune destruction of the bile ducts that transport bile acids outof the liver. As the disease progresses, persistent toxic build-up ofbile acids causes progressive liver damage marked by chronicinflammation and fibrosis. Because patients with PBC have an increasedrisk of HCC, therapy with the variants of FGF19 peptide sequences,fusions of FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences described herein isof particular import, as such sequences do not induce, or do notsubstantially increase, HCC formation or HCC tumorigenesis.

Although patients with PBC are often asymptomatic at the time of initialdiagnosis, most present, or subsequently develop, one or more of thefollowing: pruritus; fatigue; jaundice; xanthoma; disorders associatedwith an extrahepatic autoimmune disorder (e.g., Sjögren's Syndrome andrheumatoid arthritis); and complications that result from cirrhosis orportal hypertension (e.g., ascites, esophageal varices and hepaticencephalopathy).

While a definitive cause of PBC has not been identified, most researchsuggests that it is an autoimmune disorder. There appears to be agenetic predisposition, and genetic studies have indicated that part ofthe IL-12 signaling cascade, including IL-12A and I-12RB2 polymorphisms,is important in the etiology of the disease.

There is no definitive means of diagnosing PBC; rather, assessment of anumber of factors is generally required. Moreover, diagnosis of PBCrequires that other conditions with similar symptoms (e.g., autoimmunehepatitis and primary sclerosing cholangitis) by ruled out; by way ofexample, abdominal ultrasound or CT scan is usually performed to ruleout blockage of the bile ducts.

Diagnostic blood tests include deranged liver function tests(gamma-glutamyl transferase and alkaline phosphatase) and the presenceof particular antibodies (antimitochondrial antibody (AMA) anantinuclear antibody (ANA)). Antinuclear antibodies are believed to beprognostic indicators of PBC. When other tests and procedures areindicative of PBC, a liver biopsy is frequently performed to confirmdisease. Endoscopic retrograde cholangiopancreatography (ERCP), anendoscopic evaluation of the bile duct, may also be employed to confirmdisease.

PBC is classified into four stages marking the progression of disease.Stage 1 (Portal Stage) is characterized by portal inflammation and mildbile duct damage; Stage 2 (Periportal Stage) is characterized byenlarged triads, periportal fibrosis or inflammation; Stage 3 (SeptalStage) is characterized by active and/or passive fibrous septa; andStage 4 (Biliary Cirrhosis) is characterized by the presence of hepaticnodules. Liver biopsy is required to determine the stage of disease.

Serum bilirubin is an indicator of PBC progression and prognosis.Patients with a serum bilirubin level of 2-6 mg/dL have a mean survivaltime of 4.1 years, patients with a serum bilirubin level of 6-10 mg/dLhave a mean survival time of 2.1 years, and patients with a serumbilirubin level above 10 mg/dL have a mean survival time of 1.4 years.Liver transplantation is an option in advanced cases of PBC, althoughthe recurrence rate may be as high as 18% at 5 years, and up to 30% at10 years.

Although disease progression may be slowed, pharmaceutical interventionwith currently used therapies is neither curative nor effective in allpatient populations. In order to improve the therapeutic outcome ofpharmacological therapy, one aspect pertains to the use of one or morecurrent therapies in combination with variants of FGF19 peptidesequences, fusions of FGF19 and/or FGF21 peptide sequences and variantsof fusions (chimeras) of FGF19 and/or FGF21 peptide sequences having oneor more activities associated with the treatment and/or prevention ofPBC and associated diseases, disorders and conditions. The most commonlyused and/or promising agents for combination therapy are set forthhereafter, although it is to be understood that these agents areillustrative, and not exclusionary.

PBC treatment most frequently involves the bile acid ursodeoxycholicacid (Urosdiol, UDCA). UDCA therapy is helpful in reducing thecholestasis and improving the liver function tests in PBC patients;however, it does not demonstrably improve symptoms and has aquestionable impact on prognosis. UDCA has been shown to reducemortality, adverse events and the need for transplantation in PBC.Although UDCA is considered the first-line therapy, approximatelyone-third of patients may be non-responsive and remain at risk ofprogressive liver disease and are candidates for alternative or additivetherapy.

There are several alternative and adjuvant therapies, some of which arecurrently in clinical development, that can be used in combination withvariants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21peptide sequences and variants of fusions (chimeras) of FGF19 and/orFGF21 peptide sequences provided herein having one or more activitiesassociated with the treatment and/or prevention of PBC and associateddiseases, disorders and conditions.

Farnesoid-X-receptor agonists represent a promising class of agents thatmay be used in combination therapy. One of the primary functions ofagonists of FXR, a nuclear receptor expressed at high levels in theliver and intestine, is the suppression of cholesterol 7α hydroxylase-1(CYP7A1), the rate-limiting enzyme in the synthesis of bile acids fromcholesterol. Obeticholic acid (OCA; Intercept Pharmaceuticals, NY) is abile acid analog and FXR agonist derived from the primary human bileacid chenodeoxycholic acid, or CDCA. OCA is currently being evaluatedfor patients having an inadequate therapeutic response to ursodiol orwho are unable to tolerate ursodiol.

Inhibitors of the apical sodium-dependent bile acid transporter (ASBT)represent another class of agents that may be used in combination withthe variants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21peptide sequences and variants of fusions (chimeras) of FGF19 and/orFGF21 peptide sequences described herein for the treatment and/orprevention of PBC and associated diseases. ASBT, a member of thesodium/bile-salt co-transport family coded by gene SLC10A2, is currentlythought to be the primary mechanism for bile acid reabsorption in theintestine. Examples of ABST inhibitors include LUM001 and SC-435, bothof which are being developed by Lumena Pharmaceuticals (San Diego,Calif.).

Bile acid sequestrants also find use in the treatment of PBC.Cholestyramine and colestipol are the best known bile acid sequestrants.However, their use is sometimes limited because they are only availablein powder form and are not tolerated by many patients, often because ofthe poor texture and taste of the resin powder. The bile acidsequestrant colesevelam is available in tablet form and is often bettertolerated. All bile acid sequestrants are capable of binding othercompounds, including the fat-soluble vitamins A, D, E and K, anddeficiencies of these vitamins many necessitate supplementation.Importantly, the PBC patient population inherently has poorlipid-dependent absorption of vitamins A, D, E and K, and thus patientstaking bile acid sequestrants are at particular risk for deficiency ofthose vitamins.

Agents associated with immune and inflammatory function are candidatesfor combination therapy with the variants of FGF19 peptide sequences,fusions of FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences having one or moreactivities associated with the treatment and/or prevention of PBC andassociated diseases, disorders and conditions.

The interleukin IL-12 is linked with autoimmunity. Data indicate thatthe IL-12 signaling pathway plays a key role in the effector mechanismsthat lead to biliary destruction. Targeting the p40 subunit of IL-12 hasalso been shown to ameliorate experimental immune-mediatedcholangiopathy. Thus, anti-IL-12 agents (e.g., monoclonal Ab inhibitors)provide a promising treatment. Furthermore, because polymorphisms inCD80 have been identified as conferring an increased susceptibility toPBC, blockade of co-stimulation between T cells and antigen-presentingcells through CD80 by use of an anti-CD80 agent could represent animportant therapeutic approach for the treatment of PBC. In addition,improvement in IgM titre and an increase in intrahepatic regulatoryT-cell number using the anti-CD20 antibody rituximab (RITUXAN) haveshown promise.

The immune-mediated destruction of small-sized bile ducts in PBC ispredominantly cell-mediated, characterized by Th1 cells, CD8+ T cells,NK cells and NKT cells which express CXCR3. Therefore, neutralizingantibodies to CXCL10, a ligand for CXCR3, may offer the possibility tointerfere with one of the key inflammatory processes and contribute toimmune-mediated biliary destruction in PBC. Similarly, blockade ofco-stimulatory signals between T cells expressing CD28 andantigen-presenting cells expressing CD80 (e.g. cholangiocytes,antibody-secreting B cells) might represent an important approach forthe treatment of autoimmune diseases.

The variants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21peptide sequences and variants of fusions (chimeras) of FGF19 and/orFGF21 peptide sequences described herein can be used alone or incombination with other agents for the treatment and/or prevention ofthose bile acid-related or associated disorders referenced herein thathave an immune and/or inflammatory component, including, but not limitedto, PBC and associated diseases, disorders and conditions. Examples ofsuch other agents include, for example, non-steroidal anti-inflammatorydrugs (NSAID); steroids; cytokine suppressive anti-inflammatory drug(s)(CSAIDs); antibodies to, or antagonists of, other human cytokines orgrowth factors (e.g., IL-2, IL-6, or PDGF); TNF antagonists (e.g.,agents such as REMICADE, p75TNFRIgG (ENBREL) or p55TNFR1gG (LENERCEPT));interferon-β1a (AVONEX); interferon-β1b (BETASERON); and immunecheckpoint inhibitors, including PD1 (associated agents include theantibodies nivolumab and lambrolizumab), PDL1, BTLA, CTLA4 (associatedagents include the fully humanized CTLA4 monoclonal antibody ipilimumab(YERVOY), TIM3, LAG3, and A2aR.

Fibrates have been shown to improve various aspects of PBC, includingliver function tests, both as monotherapy and in combination with UDCAnon-responders. In certain embodiments, a fibrate is a member selectedfrom the group of bezafibrate (BEZALIP), ciprofibrate (MODALIM),gemfibrozil (LOPID), clofibrate, and fenofibrate (TRICOR). Fish oil hasexhibited similar benefits.

In PBC patients demonstrating certain characteristics of hepatitis onbiopsy, corticosteroids such as budesonide may improve liver histologyand biochemistry, particularly when used in combination with UDCA.Colchicine has been shown to improve liver function tests (e.g., AST andALP) and represents another alternative treatment for PBC.

Though not an exhaustive list, other drugs that have shown promiseinclude methotrexate as an immunomodulatory treatment, azathioprine,cyclosporine, and certain agents used in anti-retroviral therapy (e.g.,combivir).

Various treatments exist for the sequelae associated with PBC. Forexample, itching can be relieved by the bile acid sequestrantcholestyramine, or alternatively naltrexone and rifampicin. The fatigueassociated with PBC may effectively be treated with modafinil (Provigil;Teva (formerly Cephalon)) without damaging the liver. As patients withPBC have increased risk of developing osteoporosis and esophagealvarices compared to the general population (and others with liverdisease), screening and treatment of these complications is an importantpart of the management of PBC. Variants of FGF19 peptide sequences,fusions of FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences having one or moreactivities associated with the treatment and/or prevention of PBC andassociated diseases, disorders and conditions, as provided herein,either alone or in combination with other agents, offer novel, promisingalternatives to the management of such sequelae.

5.6.1.2 NASH and NAFLD and Therapy with Agents Effective in theTreatment or Prevention Thereof

Non-alcoholic steatohepatitis (NASH), considered part of a spectrum ofnon-alcoholic fatty liver diseases (NAFLD), causes inflammation andaccumulation of fat and fibrous tissue in the liver. Although the exactcause of NASH is unknown, risk factors include central obesity, type-2diabetes mellitus, insulin resistance (IR) and dyslipidemia;combinations of the foregoing are frequently described as the metabolicsyndrome. In addition, certain drugs have been linked to NASH, includingtamoxifen, amiodarone and steroids (e.g., prednisone andhydrocortisone). Non-alcoholic fatty liver disease is the most commoncause of chronic liver disease in the United States, and the estimatedprevalence of NAFLD is 20-30% and for NASH it is estimated at 3.5-5%.(See, e.g., Abrams, G. A., et al., Hepatology, 2004. 40(2):475-83;Moreira, R. K., Arch Pathol Lab Med, 2007. 131(11):1728-34).

NASH frequently presents with no overt symptoms, complicating itsdiagnosis. Liver function tests generally begin the diagnostic process,with levels of AST (aspartate aminotransferase) and ALT (alanineaminotransferase) elevated in about 90% percent of individuals withNASH. Other blood tests are often used for ruling out other causes ofliver disease, such as hepatitis. Imaging tests (e.g., ultrasound, CTscan, or MRI) may reveal fat accumulation in the liver but frequentlycannot differentiate NASH from other causes of liver disease that have asimilar appearance. A liver biopsy is required to confirm NASH.

The prognosis for individuals suffering from NASH is difficult topredict, although features in the liver biopsy can be helpful. The mostserious complication of NASH is cirrhosis, which occurs when the liverbecomes severely scarred. It has been reported that between 8 and 26percent of individuals with NASH develop cirrhosis, and it is predictedthat NASH will be the leading indication for liver transplantation by2020.

At the present time, treatment of NASH focuses primarily onpharmacological and non-pharmacological management of those medicalconditions associated with it, including hyperlipidemia, diabetes andobesity. Although not curative, pharmacological intervention of NASHitself includes treatment with vitamin E, pioglitazone, metformin,statins, omega-3 fatty acids, and ursodeoxycholic acid (UDCA(ursodiol)). Other agents being evaluated, currently approved fordifferent indications, include losartan and telisartan, exenatide, GLP-1agonists, DPP IV inhibitors, and carbamazepine.

In view of the deficiencies of the aforementioned current therapies,therapy with agents having distinct mechanisms of action offers apromising new avenue for the treatment and prevention of NASH and NAFLD.Addressing such deficiencies is contemplated, for example, by using thevariants of FGF19 peptide sequences, fusions of FGF19 and/or FGF21peptide sequences and variants of fusions (chimeras) of FGF19 and/orFGF21 peptide sequences as taught herein. In certain embodiments, thepeptides are used in combination with other therapeutic agents and/ortreatment modalities. Also provided herein is the prophylactic and/ortherapeutic use of these variants of FGF19 peptide sequences, fusions ofFGF19 and/or FGF21 peptide sequences and variants of fusions (chimeras)of FGF19 and/or FGF21 peptide sequences, either alone or in combinationwith therapies developed in the future, for the treatment or preventionof NASH and NAFLD.

5.6.1.3 Therapy for the Treatment or Prevention of Other BileAcid-Related Disorders and Associated Diseases, Disorders and Conditions

Also provided herein is the use of variants of FGF19 peptide sequences,fusions of FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences having one or moreactivities associated with the treatment and/or prevention of other bileacid-related disorders and associated diseases, disorders and conditionsbesides PBC. In certain embodiments, the peptides are used incombination with other therapeutic agents and/or treatment modalities.

By way of example, patients with bile acid diarrhea secondary to Crohn'sileitis will be helped with glucocorticoid treatment. Microscopiccolitis is also helped by steroids. In patients with a short-bowelsyndrome (a bile acid deficiency occurs in the proximal intestine thatleads to impaired micellar solubilization), cholylsarcosine(cholyl-N-methylglycine), a synthetic bile acid analogue, has been shownto increase lipid absorption.

Administration of the primary bile acid chenodeoxycholic Acid (CDCA) hasbeen shown to decrease biliary cholesterol secretion and gradualdissolution of gallstones. Because CDCA is slightly hepatotoxic, it wasgradually replaced by UDCA. Despite the efficacy and safety of UDCAadministration for cholesterol gallstone dissolution, it is notfrequently used today because of the success of laparoscopiccholecystectomy, which provides a rapid cure for symptomatic disease.Medical therapy, in contrast, requires months of therapy, does notalways dissolve stones, and is followed by gradual recurrence in somepatients.

Bile acid replacement is used in inborn errors of bile acidbiosynthesis, usually with a mixture of CDCA or UDCA and cholic acid, tosuppress the synthesis of cytotoxic bile acid precursors and restore theinput of primary bile acids into the enterohepatic circulation.

In addition to the agents and therapeutic modalities set forth above,combination therapy with numerous additional agents (and classesthereof) is also contemplated, including. but not limited to, 1) insuline.g., bolus and basal analogs), insulin mimetics and agents that entailstimulation of insulin secretion, including sulfonylureas (e.g.,chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide,glimepiride, glipizide) and meglitinides (e.g., repaglinide (PRANDIN)and nateglinide (STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE))and other agents that act by promoting glucose utilization, reducinghepatic glucose production and/or diminishing intestinal glucose output;3) alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and otheragents that slow down carbohydrate digestion and consequently absorptionfrom the gut and reduce postprandial hyperglycemia; 4)thiazolidinediones (e.g., rosiglitazone (AVANDIA), troglitazone(REZULIN), pioglitazone (ACTOS), glipizide, balaglitazone,rivoglitazone, netoglitazone, troglitazone, englitazone, ciglitazone,adaglitazone, darglitazone that enhance insulin action (e.g., by insulinsensitization), thus promoting glucose utilization in peripheraltissues; 5) glucagon-like-peptides including DPP-IV inhibitors (e.g.,vildagliptin (GALVUS) and sitagliptin (JANUVIA)) and Glucagon-LikePeptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g., exenatide(BYETTA and ITCA 650 (an osmotic pump inserted subcutaneously thatdelivers an exenatide analog over a 12-month period; Intarcia, Boston,Mass.)); 6) and DPP-IV-resistant analogues (incretin mimetics), PPARgamma agonists, dual-acting PPAR agonists, pan-acting PPAR agonists,PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXR agonists,glycogen synthase kinase-3 inhibitors, immune modulators, beta-3adrenergic receptor agonists, 11beta-HSD1 inhibitors, and amylinanalogues.

Other exemplary agents that can be used, in certain embodiments, incombination with the peptides and methods provided herein includedipeptidyl peptidase-4 (DPP-4) inhibitors, bromocriptine formulations(e.g. and bile acid sequestrants (e.g., colesevelam), and SGLT-2inhibitors. Appetite suppression drugs are also well known and can beused in combination with the compositions and methods provided herein.Supplementary therapies can be administered prior to, contemporaneouslywith or following methods and uses provided herein

5.6.2 Methods of Preventing, Treating and Managing Metabolic Disorders

Also provided herein are in vitro, ex vivo and in vivo (e.g., on or in asubject) methods and uses. Such methods and uses can be practiced withany of the peptide sequences set forth herein. In various embodiments,the methods include administering a peptide sequence, such as a FGF19 orFGF21 variant, fusion or chimera disclosed herein (e.g., in the SequenceListing or Table 1), or a subsequence, a variant or modified form of aFGF19 or FGF21 variant, fusion or chimera disclosed herein (e.g., theSequence Listing or Table 1), to a subject in an amount effective fortreating a metabolic or associated disorder.

In certain embodiments, the peptide is administered in combination withan additional therapeutic agent(s) and/or treatment modalities (e.g., anagent useful in the treatment and/or prevention of PBC). The additionaltherapeutic agent(s) can be administered before, with, or followingadministration of the peptides described herein.

Also provided here are methods of preventing (e.g., in subjectspredisposed to having a particular disorder(s)), delaying, slowing orinhibiting progression of, the onset of, or treating (e.g.,ameliorating) a metabolic or associated disorder relative to anappropriate matched subject of comparable age, gender, race, etc.).Thus, in various embodiments, a method provided herein for, for example,modulating bile acid homeostasis or treating a metabolic or associateddisorder includes contacting or administering one or more peptidesprovided herein (e.g., a variant or fusion of FGF19 and/or FGF21 as setforth in the Sequence Listing or Table 1) in an amount effective tomodulate bile acid homeostasis or treat a metabolic or associateddisorder. In certain embodiments the method further comprises contactingor administering at least one additional therapeutic agent or treatmentmodality that is useful in the treatment or prevention of a metabolic orassociated disorder (e.g., PBC).

The term “subject” refers to an animal. Typically, the animal is amammal that would benefit from treatment with a peptide sequenceprovided herein. Particular examples include primates (e.g., humans),dogs, cats, horses, cows, pigs, and sheep.

Subjects include those having a disorder, e.g., a metabolic orassociated disorder, or subjects that do not have a disorder but may beat risk of developing the disorder.

Non-limiting exemplary disorders or conditions preventable, treatable ormanageable with the peptide formulations, methods and uses thereofprovided herein, include metabolic diseases and disorders. Non limitingexamples of diseases and disorders include: metabolic syndrome; a lipid-or glucose-related disorder; cholesterol or triglyceride metabolism;type 2 diabetes; cholestasis, including, for example diseases ofintrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC, neonatalcholestasis, and drug induced cholestasis (e.g., estrogen)), anddiseases of extrahepatic cholestasis (e.g., bile cut compression fromtumor, bile duct blockade by gall stones); bile acid malabsorption andother disorders involving the distal small intestine, including ilealresection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension. For treatment, peptide sequences provided herein can beadministered to subjects in need of modulation of bile acid homeostasisor having a bile-acid related or associated disorder. Peptide sequencesprovided herein may also be useful in other hyperglycemic-relateddisorders, including kidney damage (e.g., tubule damage or nephropathy),liver degeneration, eye damage (e.g., diabetic retinopathy orcataracts), and diabetic foot disorders; dyslipidemias and theirsequelae such as, for example, atherosclerosis, coronary artery disease,cerebrovascular disorders and the like.

Other conditions which may be associated with metabolic syndrome, suchas obesity and elevated body mass (including the co-morbid conditionsthereof such as, but not limited to, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovariansyndrome (PCOS)), and also include thromboses, hypercoagulable andprothrombotic states (arterial and venous), hypertension (includingportal hypertension (defined as a hepatic venous pressure gradient(HVPG) greater than 5 mm Hg), cardiovascular disease, stroke and heartfailure; Disorders or conditions in which inflammatory reactions areinvolved, including atherosclerosis, chronic inflammatory bowel diseases(e.g., Crohn's disease and ulcerative colitis), asthma, lupuserythematosus, arthritis, or other inflammatory rheumatic disorders;Disorders of cell cycle or cell differentiation processes such asadipose cell tumors, lipomatous carcinomas including, for example,liposarcomas, solid tumors, and neoplasms; Neurodegenerative diseasesand/or demyelinating disorders of the central and peripheral nervoussystems and/or neurological diseases involving neuroinflammatoryprocesses and/or other peripheral neuropathies, including Alzheimer'sdisease, multiple sclerosis, Parkinson's disease, progressive multifocalleukoencephalopathy and Guillian-Barre syndrome; Skin and dermatologicaldisorders and/or disorders of wound healing processes, includingerythemato-squamous dermatoses; and other disorders such as syndrome X,osteoarthritis, and acute respiratory distress syndrome.

In one embodiment, a subject has a hyperglycemic condition (e.g.,diabetes, such as insulin-dependent (type I) diabetes, type II diabetes,or gestational diabetes), insulin resistance, hyperinsulinemia, glucoseintolerance or metabolic syndrome, is obese and/or has an undesirablebody mass.

In particular aspects of the methods and uses, a peptide sequence orchimeric peptide sequence provided herein is administered to a subjectin an amount effective to improve glucose metabolism in the subject. Inmore particular aspects, a subject has a fasting plasma glucose levelgreater than 100 mg/dl or has a hemoglobin A1c (HbA1c) level above 6%,prior to administration.

In further embodiments, a use or method of treatment of a subject isintended to or results in reduced glucose levels, increased insulinsensitivity, reduced insulin resistance, reduced glucagon, animprovement in glucose tolerance, or glucose metabolism or homeostasis,improved pancreatic function, or reduced triglyceride, cholesterol, IDL,LDL or VLDL levels, or a decrease in blood pressure, a decrease inintimal thickening of the blood vessel, or a decrease in body mass orweight gain.

Treatment of a metabolic or associated disorder (e.g., hyperglycemia)may have the benefit of alleviating or abolishing a disorder secondarythereto. By way of example, a subject suffering from hyperglycemia mayalso have depression or anxiety due to the hyperglycemia; thus, treatingthe subject's hyperglycemia may also indirectly treat the depression oranxiety. The use of the therapies disclosed herein to target suchsecondary disorders is also contemplated in certain embodiments.

In particular embodiments, the subject has or is at risk of havinghyperglycemia. In other particular embodiments, the subject has or is atrisk of having diabetes, such as Type 2 diabetes.

Subjects at risk of developing a metabolic or associated disorder (suchas the disorders described above) include, for example, those who mayhave a family history or genetic predisposition toward such disorder, aswell those whose diet may contribute to development of such disorders.

As disclosed herein, treatment methods include contacting oradministering a peptide as set forth herein (e.g., a variant or fusionof FGF19 and/or FGF21 as set forth in the Sequence Listing or Table 1)in an amount effective to achieve a desired outcome or result in asubject. A treatment that results in a desired outcome or resultincludes decreasing, reducing or preventing the severity or frequency ofone or more symptoms of the condition in the subject, e.g., animprovement in the subject's condition or a “beneficial effect” or“therapeutic effect.” Therefore, treatment can decrease or reduce orprevent the severity or frequency of one or more symptoms of thedisorder, stabilize or inhibit progression or worsening of the disorder,and in some instances, reverse the disorder, transiently (e.g., for 1-6,6-12, or 12-24 hours), for medium term (e.g., 1-6, 6-12, 12-24 or 24-48days) or long term (e.g., for 1-6, 6-12, 12-24, 24-48 weeks, or greaterthan 24-48 weeks). Thus, in the case of a metabolic or associateddisorder, treatment can lower or reduce one or more symptoms or effectsof the metabolic or associated disorders described above.

In certain embodiments, the various methods provided herein furtherinclude contacting or administering one or more additional agents ortherapeutic modalities useful in the treatment or prevention of ametabolic or associated disorder, such as those agents or therapeuticmodalities described herein, in an amount effective to achieve a desiredoutcome or result in a subject.

An “effective amount” or a “sufficient amount” for use and/or fortreating a subject refers to an amount that provides, in single ormultiple doses, alone, or in combination with one or more other agents,treatments, protocols, or therapeutic regimens, a detectable response ofany duration of time (transient, medium or long term), a desired outcomein or an objective or subjective benefit to a subject of any measurableor detectable degree or for any duration of time (e.g., for hours, days,months, years, in remission or cured). Such amounts typically areeffective to ameliorate a disorder, or one, multiple or all adversesymptoms, consequences or complications of the disorder, to a measurableextent, although reducing or inhibiting a progression or worsening ofthe disorder, is considered a satisfactory outcome.

As used herein, the term “ameliorate” means an improvement in thesubject's disorder, a reduction in the severity of the disorder, or aninhibition of progression or worsening of the disorder (e.g.,stabilizing the disorder). In the case of a metabolic or associateddisorder such as those described above, an improvement can be a loweringor a reduction in one or more symptoms or effects of the disorder.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the disorder or disease. Thus, asatisfactory endpoint is achieved when there is a transient, medium orlong term, incremental improvement in a subject's condition, or apartial reduction in the occurrence, frequency, severity, progression,or duration, or inhibition or reversal, of one or more associatedadverse symptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the condition, disorder or disease), of the disorder ordisease, over a duration of time (hours, days, weeks, months, etc.).

Thus, in the case of a disorder treatable by a peptide sequence providedherein, either alone or in combination with an additional agent, theamount of the peptide (and optionally the additional agent) sufficientto ameliorate a disorder will depend on the type, severity and extent,or duration of the disorder, the therapeutic effect or outcome desired,and can be readily ascertained by the skilled artisan. Appropriateamounts will also depend upon the individual subject (e.g., thebioavailability within the subject, gender, age, etc.). For example, atransient, or partial, restoration of normal bile acid homeostasis in asubject can reduce the dosage amount or frequency of the peptides andagents described herein in order to treat the metabolic or associateddisorders described previously even though complete freedom fromtreatment has not resulted. An effective amount can be ascertained, forexample, by measuring one or more relevant physiological effects.

Methods and uses provided herein for treating a subject are applicablefor prophylaxis to prevent or reduce the likelihood of a disorder in asubject, such as a metabolic or associated disorder. Accordingly,methods and uses provided herein for treating a subject having, or atrisk of developing, a metabolic or associated disorder can be practicedprior to, substantially contemporaneously with, or followingadministration or application of another agent useful for the treatmentor prevention of a metabolic or associated disorder, and/or can besupplemented with other forms of therapy. Supplementary therapiesinclude other glucose lowering treatments, such as insulin, an insulinsensitivity enhancer and other drug treatments, a change in diet (lowsugar, fats, etc.), weight loss surgery- (reducing stomach volume bygastric bypass, gastrectomy), gastric banding, gastric balloon, gastricsleeve, etc. For example, a method or use provided herein for treating ahyperglycemic or insulin resistance disorder can be used in combinationwith drugs or other pharmaceutical compositions that lower glucose orincrease insulin sensitivity in a subject.

In one embodiment, a method or use includes contacting or administeringto a subject one or more variant or fusion FGF19 and/or FGF21 peptidesequences in an amount effective for preventing a metabolic orassociated disorder. In one embodiment, a method or use includescontacting or administering to a subject one or more variant or fusionFGF19 and/or FGF21 peptide sequences in an amount effective for treatinga metabolic or associated disorder. In one embodiment, a method or useincludes contacting or administering to a subject one or more variant orfusion FGF19 and/or FGF21 peptide sequences in an amount effective formanaging a metabolic or associated disorder.

5.6.3 Methods of Preventing, Treating and Managing Cancer

Also provided herein are in vitro, ex vivo and in vivo (e.g., on or in asubject) methods and uses. Such methods and uses can be practiced withany of the peptide sequences set forth herein. In various embodiments,the methods include administering a peptide sequence, such as a FGF19 orFGF21 variant, fusion or chimera disclosed herein (e.g., in the SequenceListing or Table 1), or a subsequence, a variant or modified form of aFGF19 or FGF21 variant, fusion or chimera disclosed herein (e.g., theSequence Listing or Table 1), to a subject in an amount effective fortreating a cancer, tumor or associated disorder.

In certain embodiments, the peptide is administered in combination withan additional therapeutic agent(s) and/or treatment modalities (e.g., anagent useful in the treatment and/or prevention of PBC). The additionaltherapeutic agent(s) can be administered before, with, or followingadministration of the peptides described herein.

Also provided here are methods of preventing (e.g., in subjectspredisposed to having a particular disorder(s)), delaying, slowing orinhibiting progression of, the onset of, or treating (e.g.,ameliorating) a cancer, tumor or associated disorder relative to anappropriate matched subject of comparable age, gender, race, etc.).Thus, in various embodiments, a method provided herein for, for example,preventing or treating a cancer, tumor or associated disorder includescontacting or administering one or more peptides provided herein (e.g.,a variant or fusion of FGF19 and/or FGF21 as set forth in the SequenceListing or Table 1) in an amount effective to prevent or treat a cancer,tumor or associated disorder. In certain embodiments the method furthercomprises contacting or administering at least one additionaltherapeutic agent or treatment modality that is useful in the treatmentor prevention of a cancer, tumor or associated disorder (e.g., PBC).

The term “subject” refers to an animal. Typically, the animal is amammal that would benefit from treatment with a peptide sequenceprovided herein. Particular examples include primates (e.g., humans),dogs, cats, horses, cows, pigs, and sheep.

Subjects include those having a disorder, e.g., a cancer, tumor orassociated disorder; or subjects that do not have a disorder but may beat risk of developing the disorder.

Non-limiting exemplary disorders or conditions preventable, treatable ormanageable with the peptide formulations, methods and uses thereofprovided herein, include cancer, tumors and associated diseases anddisorders. Non limiting examples of diseases and disorders include:

In certain embodiments, the cancer or tumor is a colon tumor or ahepatic tumor.

Treatment of a cancer, tumor or associated disorder may have the benefitof alleviating or abolishing a disorder secondary thereto. By way ofexample, a subject suffering from a cancer or tumor may also havedepression or anxiety due to the cancer or tumor; thus, treating thesubject's cancer or tumor may also indirectly treat the depression oranxiety. The use of the therapies disclosed herein to target suchsecondary disorders is also contemplated in certain embodiments.

Subjects at risk of developing a cancer, tumor or associated disorder(such as the disorders described above) include, for example, those whomay have a family history or genetic predisposition toward suchdisorder, as well those whose diet may contribute to development of suchdisorders.

In some embodiments, the cancer or tumor is a liver, colon, prostate orlung cancer or tumor. In some embodiments, the cancer or tumor isbenign. In other embodiments, the cancer or tumor is malignant.

In certain embodiments, the subject has or is at risk of developing aFGF19-dependent disease, disorder or condition. In some embodiments, theFGF19-dependent disease, disorder or condition is a liver(hepatocellular) disease, disorder or condition, such as cirrhosis orcholestasis. In some embodiments, the liver disease or disorder is achronic liver disease or disorder. In some embodiments, theFGF19-dependent disease, disorder or condition is cancer or tumor, suchas HCC. In other embodiments, the FGF19-dependent disease, disorder orcondition is not a liver disease, disorder or condition, such ascirrhosis or cholestasis. In some embodiments, the FGF19-dependentdisease, disorder or condition is not a cancer or tumor, such as HCC. Insome embodiments, the FGF19-dependent disease, disorder or condition isa colon cancer or tumor. In certain embodiments, the colon cancer ortumor is a colon adenocarcinoma. In some embodiments, theFGF19-dependent disease, disorder or condition is a prostate cancer ortumor. In yet other embodiments, the FGF19-dependent disease, disorderor condition is a lung cancer or tumor. In certain embodiments, the lungcancer or tumor is a lung squamous cell carcinoma. In some embodiments,FGF19 is expressed in a primary or metastatic cancer or tumor cell. Incertain embodiments, the FGF19-dependent disease, disorder or conditionis pre-cancerous. For example, cirrhosis and cholestasis sometimes tolead to liver cancers, such as HCC, and methods of treating orpreventing such liver diseases and disorders are contemplated. Incertain embodiments, the subject is a subject in need of prevention ortreatment thereof. In some embodiments, administration of the FGF19variant maintains bile acid homeostasis in the subject.

Also provided herein is a method of treating a cancer or tumor, such asa FGF19-dependent cancer or tumor, or a symptom thereof, in a subject,comprising administering to the subject a therapeutically effectiveamount of a FGF19 variant. In certain embodiments, the administrationresults in an improvement in the cancer, tumor or symptom thereof in thesubject. In some embodiments, the method results in a reduction in tumornumber, tumor size, or tumor weight. Also provided herein is a method ofpreventing a cancer or tumor, such as a FGF19-dependent cancer or tumor,or a symptom thereof, in a subject, comprising administering to thesubject a therapeutically effective amount of a FGF19 variant. In someembodiments, the administration results in prevention of the cancer,tumor, or symptom thereof in the subject. In some embodiments, themethod results in a reduction in tumor number, tumor size, or tumorweight. In a specific embodiment, the cancer or tumor is aFGF19-dependent cancer or tumor. In certain embodiments, the cancer ortumor is hepatocellular carcinoma. In some embodiments, the cancer ortumor is not hepatocellular carcinoma. In one embodiment, the cancer ortumor is a colon cancer or tumor. In some embodiments, the cancer ortumor is a prostate cancer or tumor. In certain embodiments, the canceror tumor is a lung cancer or tumor. In certain embodiments, the FGF19variant is a polypeptide comprising an amino acid sequence set forth inSEQ ID NO:70. In some embodiments, the FGF19 variant is a polypeptideconsisting of an amino acid sequence set forth in SEQ ID NO:70. Incertain embodiments, the subject is a subject in need thereof.

As disclosed herein, treatment methods include contacting oradministering a peptide as set forth herein (e.g., a variant or fusionof FGF19 and/or FGF21 as set forth in the Sequence Listing or Table 1)in an amount effective to achieve a desired outcome or result in asubject. A treatment that results in a desired outcome or resultincludes decreasing, reducing or preventing the severity or frequency ofone or more symptoms of the condition in the subject, e.g., animprovement in the subject's condition or a “beneficial effect” or“therapeutic effect.” Therefore, treatment can decrease or reduce orprevent the severity or frequency of one or more symptoms of thedisorder, stabilize or inhibit progression or worsening of the disorder,and in some instances, reverse the disorder, transiently (e.g., for 1-6,6-12, or 12-24 hours), for medium term (e.g., 1-6, 6-12, 12-24 or 24-48days) or long term (e.g., for 1-6, 6-12, 12-24, 24-48 weeks, or greaterthan 24-48 weeks). Thus, in the case of a cancer, tumor or associateddisorder, treatment can lower or reduce one or more symptoms or effectsof the cancer, tumor or associated disorders described above.

In certain embodiments, the various methods provided herein furtherinclude contacting or administering one or more additional agents ortherapeutic modalities useful in the treatment or prevention of acancer, tumor or associated disorder, such as those agents ortherapeutic modalities described herein, in an amount effective toachieve a desired outcome or result in a subject.

An “effective amount” or a “sufficient amount” for use and/or fortreating a subject refers to an amount that provides, in single ormultiple doses, alone, or in combination with one or more other agents,treatments, protocols, or therapeutic regimens, a detectable response ofany duration of time (transient, medium or long term), a desired outcomein or an objective or subjective benefit to a subject of any measurableor detectable degree or for any duration of time (e.g., for hours, days,months, years, in remission or cured). Such amounts typically areeffective to ameliorate a disorder, or one, multiple or all adversesymptoms, consequences or complications of the disorder, to a measurableextent, although reducing or inhibiting a progression or worsening ofthe disorder, is considered a satisfactory outcome.

As used herein, the term “ameliorate” means an improvement in thesubject's disorder, a reduction in the severity of the disorder, or aninhibition of progression or worsening of the disorder (e.g.,stabilizing the disorder). In the case of a cancer, tumor or associateddisorder such as those described above, an improvement can be a loweringor a reduction in one or more symptoms or effects of the disorder.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the disorder or disease. Thus, asatisfactory endpoint is achieved when there is a transient, medium orlong term, incremental improvement in a subject's condition, or apartial reduction in the occurrence, frequency, severity, progression,or duration, or inhibition or reversal, of one or more associatedadverse symptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the condition, disorder or disease), of the disorder ordisease, over a duration of time (hours, days, weeks, months, etc.).

Thus, in the case of a disorder treatable by a peptide sequence providedherein, either alone or in combination with an additional agent, theamount of the peptide (and optionally the additional agent)sufficient toameliorate a disorder will depend on the type, severity and extent, orduration of the disorder, the therapeutic effect or outcome desired, andcan be readily ascertained by the skilled artisan. Appropriate amountswill also depend upon the individual subject (e.g., the bioavailabilitywithin the subject, gender, age, etc.). For example, a transient, orpartial, restoration of normal bile acid homeostasis in a subject canreduce the dosage amount or frequency of the peptides and agentsdescribed herein in order to treat the cancer, tumor or associateddisorders described previously even though complete freedom fromtreatment has not resulted. An effective amount can be ascertained, forexample, by measuring one or more relevant physiological effects.

Methods and uses provided herein for treating a subject are applicablefor prophylaxis to prevent or reduce the likelihood of a disorder in asubject, such as a cancer, tumor or associated disorder. Accordingly,methods and uses provided herein for treating a subject having, or atrisk of developing, a cancer, tumor or associated disorder can bepracticed prior to, substantially contemporaneously with, or followingadministration or application of another agent useful for the treatmentor prevention of a cancer, tumor or associated disorder, and/or can besupplemented with other forms of therapy. Supplementary therapiesinclude other glucose lowering treatments, such as insulin, an insulinsensitivity enhancer and other drug treatments, a change in diet (lowsugar, fats, etc.), weight loss surgery- (reducing stomach volume bygastric bypass, gastrectomy), gastric banding, gastric balloon, gastricsleeve, etc. For example, a method or use provided herein for treating ahyperglycemic or insulin resistance disorder can be used in combinationwith drugs or other pharmaceutical compositions that lower glucose orincrease insulin sensitivity in a subject.

In one embodiment, a method or use includes contacting or administeringto a subject one or more variant or fusion FGF19 and/or FGF21 peptidesequences in an amount effective for preventing a cancer, tumor orassociated disorder. In one embodiment, a method or use includescontacting or administering to a subject one or more variant or fusionFGF19 and/or FGF21 peptide sequences in an amount effective for treatinga cancer, tumor or associated disorder. In one embodiment, a method oruse includes contacting or administering to a subject one or morevariant or fusion FGF19 and/or FGF21 peptide sequences in an amounteffective for managing a cancer, tumor or associated disorder.

5.7 Nucleic Acid Molecules

Also provided are nucleic acid molecules encoding peptide sequencesprovided herein, including subsequences, sequence variants and modifiedforms of the sequences listed in the Sequence Listing (and in PCT Pub.No. WO 2013/006486 and US Pub. No. 2013/0023474, as well as PCT Publ.No. WO 2014/085365) or Table 1, and vectors that include nucleic acidencoding the peptides used in the methods described herein. Accordingly,“nucleic acids” include those that encode the exemplified peptidesequences disclosed herein, as well as those encoding functionalsubsequences, sequence variants and modified forms of the exemplifiedpeptide sequences, so long as the foregoing retain at least detectableor measurable activity or function useful in the treatment or preventionof a bile acid-related or associated disorder (e.g., PBC).

Nucleic acid, which can also be referred to herein as a gene,polynucleotide, nucleotide sequence, primer, oligonucleotide or probe,refers to natural or modified purine- and pyrimidine-containing polymersof any length, either polyribonucleotides or polydeoxyribonucleotides ormixed polyribo-polydeoxyribo nucleotides and α-anomeric forms thereof.The two or more purine- and pyrimidine-containing polymers are typicallylinked by a phosphoester bond or analog thereof. The terms can be usedinterchangeably to refer to all forms of nucleic acid, includingdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleicacids can be single strand, double, or triplex, linear or circular.Nucleic acids include genomic DNA and cDNA. RNA nucleic acid can bespliced or unspliced mRNA, rRNA, tRNA or antisense. Nucleic acidsinclude naturally occurring, synthetic, as well as nucleotide analogsand derivatives.

As a result of the degeneracy of the genetic code, the nucleic acidmolecules provided herein include sequences degenerate with respect tonucleic acid molecules encoding the peptide sequences useful in themethods provided herein. Thus, degenerate nucleic acid sequencesencoding peptide sequences, including subsequences, variants andmodified forms of the peptide sequences exemplified herein (e.g., in theSequence Listing or Table 1), are provided. The term “complementary,”when used in reference to a nucleic acid sequence, means the referencedregions are 100% complementary, i.e., exhibit 100% base pairing with nomismatches.

Nucleic acid can be produced using any of a variety of known standardcloning and chemical synthesis methods, and can be altered intentionallyby site-directed mutagenesis or other recombinant techniques known toone skilled in the art. Purity of polynucleotides can be determinedthrough, for example, sequencing, gel electrophoresis, and UVspectrometry.

Nucleic acids may be inserted into a nucleic acid construct in whichexpression of the nucleic acid is influenced or regulated by an“expression control element,” referred to herein as an “expressioncassette.” The term “expression control element” refers to one or morenucleic acid sequence elements that regulate or influence expression ofa nucleic acid sequence to which it is operatively linked. An expressioncontrol element can include, as appropriate, promoters, enhancers,transcription terminators, gene silencers, a start codon (e.g., ATG) infront of a protein-encoding gene, etc.

An expression control element operatively linked to a nucleic acidsequence controls transcription and, as appropriate, translation of thenucleic acid sequence. The term “operatively linked” refers to ajuxtaposition wherein the referenced components are in a relationshippermitting them to function in their intended manner. Typically,expression control elements are juxtaposed at the 5′ or the 3′ ends ofthe genes but can also be intronic.

Expression control elements include elements that activate transcriptionconstitutively, that are inducible (i.e., require an external signal orstimuli for activation), or derepressible (i.e., require a signal toturn transcription off; when the signal is no longer present,transcription is activated or “derepressed”). Also included in theexpression cassettes provided herein are control elements sufficient torender gene expression controllable for specific cell types or tissues(i.e., tissue-specific control elements). Typically, such elements arelocated upstream or downstream (i.e., 5′ or 3′) of the coding sequence.Promoters are generally positioned 5′ of the coding sequence. Promoters,produced by recombinant DNA or synthetic techniques, can be used toprovide for transcription of the polynucleotides provided herein. A“promoter” typically means a minimal sequence element sufficient todirect transcription.

Nucleic acids may be inserted into a plasmid for transformation into ahost cell and for subsequent expression and/or genetic manipulation. Aplasmid is a nucleic acid that can be stably propagated in a host cell;plasmids may optionally contain expression control elements in order todrive expression of the nucleic acid. As used herein, a vector issynonymous with a plasmid. Plasmids and vectors generally contain atleast an origin of replication for propagation in a cell and a promoter.Plasmids and vectors may also include an expression control element forexpression in a host cell, and are therefore useful for expressionand/or genetic manipulation of nucleic acids encoding peptide sequences,expressing peptide sequences in host cells and organisms, or producingpeptide sequences, for example.

As used herein, the term “transgene” means a polynucleotide that hasbeen introduced into a cell or organism by artifice. For example, in acell having a transgene, the transgene has been introduced by geneticmanipulation or “transformation” of the cell. A cell or progeny thereofinto which the transgene has been introduced is referred to as a“transformed cell” or “transformant.” Typically, the transgene isincluded in progeny of the transformant or becomes a part of theorganism that develops from the cell. Transgenes may be inserted intothe chromosomal DNA or maintained as a self-replicating plasmid, YAC,minichromosome, or the like.

Bacterial system promoters include T7 and inducible promoters such as pLof bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) andtetracycline-responsive promoters. Insect cell system promoters includeconstitutive or inducible promoters (e.g., ecdysone). Mammalian cellconstitutive promoters include SV40, RSV, bovine papilloma virus (BPV)and other virus promoters, or inducible promoters derived from thegenome of mammalian cells (e.g., metallothionein HA promoter; heat shockpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the inducible mouse mammary tumor virus long terminal repeat).Alternatively, a retroviral genome can be genetically modified forintroducing and directing expression of a peptide sequence inappropriate host cells.

As methods and uses provided herein include in vivo delivery, expressionsystems further include vectors designed for in vivo use. Particularnon-limiting examples include adenoviral vectors (U.S. Pat. Nos.5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat. No.5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703),BPV vectors (U.S. Pat. No. 5,719,054), CMV vectors (U.S. Pat. No.5,561,063) and parvovirus, rotavirus, Norwalk virus and lentiviralvectors (see, e.g., U.S. Pat. No. 6,013,516). Vectors include those thatdeliver genes to cells of the intestinal tract, including the stem cells(Croyle et al., Gene Ther. 5:645 (1998); S. J. Henning, Adv. Drug Deliv.Rev. 17:341 (1997), U.S. Pat. Nos. 5,821,235 and 6,110,456). Many ofthese vectors have been approved for human studies.

Yeast vectors include constitutive and inducible promoters (see, e.g.,Ausubel et al., In: Current Protocols in Molecular Biology, Vol. 2, Ch.13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al.Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; BitterMethods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad.Press, N.Y.; and, Strathern et al., The Molecular Biology of the YeastSaccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). Aconstitutive yeast promoter such as ADH or LEU2 or an inducible promotersuch as GAL may be used (R. Rothstein In: DNA Cloning, A PracticalApproach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C.,1986). Vectors that facilitate integration of foreign nucleic acidsequences into a yeast chromosome, via homologous recombination forexample, are known in the art. Yeast artificial chromosomes (YAC) aretypically used when the inserted polynucleotides are too large for moreconventional vectors (e.g., greater than about 12 Kb).

Expression vectors also can contain a selectable marker conferringresistance to a selective pressure or identifiable marker (e.g.,beta-galactosidase), thereby allowing cells having the vector to beselected for, grown and expanded. Alternatively, a selectable marker canbe on a second vector that is co-transfected into a host cell with afirst vector containing a nucleic acid encoding a peptide sequence.Selection systems include, but are not limited to, herpes simplex virusthymidine kinase gene (Wigler et al., Cell 11:223 (1977)),hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al.,Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes thatcan be employed in tk−, hgprt− or aprt− cells, respectively.Additionally, antimetabolite resistance can be used as the basis ofselection for dhfr, which confers resistance to methotrexate (O'Hare etal., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, whichconfers resistance to mycophenolic acid (Mulligan et al., Proc. Natl.Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistanceto aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1(1981)); puromycin; and hygromycin gene, which confers resistance tohygromycin (Santerre et al., Gene 30:147 (1984)). Additional selectablegenes include trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988));and ODC (ornithine decarboxylase), which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO(McConlogue (1987) In: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory).

5.8 Cell Lines and Animal Models

In certain embodiments, also provided is a transformed cell(s) (invitro, ex vivo and in vivo) and host cells that produce a variant orfusion of FGF19 and/or FGF21 as set forth herein, where expression ofthe variant or fusion of FGF19 and/or FGF21 is conferred by a nucleicacid encoding the variant or fusion of FGF19 and/or FGF21. As usedherein, a “transformed” or “host” cell is a cell into which a nucleicacid is introduced that can be propagated and/or transcribed forexpression of an encoded peptide sequence. The term also includes anyprogeny or subclones of the host cell. Transformed and host cells thatexpress peptide sequences provided herein typically include a nucleicacid that encodes the peptide sequence. In one embodiment, a transformedor host cell is a prokaryotic cell. In another embodiment, a transformedor host cell is a eukaryotic cell. In various aspects, the eukaryoticcell is a yeast or mammalian (e.g., human, primate, etc.) cell.

Transformed and host cells include but are not limited to microorganismssuch as bacteria and yeast; and plant, insect and mammalian cells. Forexample, bacteria transformed with recombinant bacteriophage nucleicacid, plasmid nucleic acid or cosmid nucleic acid expression vectors;yeast transformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); and animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered fortransient or stable propagation or expression.

For gene therapy uses and methods, a transformed cell can be in asubject. A cell in a subject can be transformed with a nucleic acid thatencodes a peptide sequence as set forth herein in vivo. Alternatively, acell can be transformed in vitro with a transgene or polynucleotide, andthen transplanted into a tissue of subject in order to effect treatment.Alternatively, a primary cell isolate or an established cell line can betransformed with a transgene or polynucleotide that encodes a variant ofFGF19 and/or FGF21 or a fusion/chimeric sequence (or variant) thereof,such as a chimeric peptide sequence including all or a portion of FGF19,or including all or a portion of FGF21, and then optionally transplantedinto a tissue of a subject.

Non-limiting target cells for expression of peptide sequences,particularly for expression in vivo, include pancreas cells (isletcells), muscle cells, mucosal cells and endocrine cells. Such endocrinecells can provide inducible production (secretion) of a variant of FGF19and/or FGF21, or a fusion/chimeric sequence (or variant) thereof, suchas a chimeric peptide sequence including all or a portion of FGF19, orincluding all or a portion of FGF21. Additional cells to transforminclude stem cells or other multipotent or pluripotent cells, forexample, progenitor cells that differentiate into the various pancreascells (islet cells), muscle cells, mucosal cells and endocrine cells.Targeting stem cells provides longer term expression of peptidesequences provided herein.

As used herein, the term “cultured,” when used in reference to a cell,means that the cell is grown in vitro. A particular example of such acell is a cell isolated from a subject, and grown or adapted for growthin tissue culture. Another example is a cell genetically manipulated invitro, and transplanted back into the same or a different subject.

The term “isolated,” when used in reference to a cell, means a cell thatis separated from its naturally occurring in vivo environment.“Cultured” and “isolated” cells may be manipulated by the hand of man,such as genetically transformed. These terms include any progeny of thecells, including progeny cells that may not be identical to the parentalcell due to mutations that occur during cell division. The terms do notinclude an entire human being.

Nucleic acids encoding peptide sequences provided herein can beintroduced for stable expression into cells of a whole organism. Suchorganisms, including non-human transgenic animals, are useful forstudying the effect of peptide expression in a whole animal andtherapeutic benefit. For example, as disclosed herein, production of avariant of FGF19 and/or FGF21 or a fusion/chimeric sequence (or variant)thereof, such as a chimeric peptide sequence including all or a portionof FGF19, or including all or a portion of FGF21 as set forth herein, inmice.

Mice strains that develop or are susceptible to developing a particulardisease (e.g., diabetes, degenerative disorders, cancer, etc.) are alsouseful for introducing therapeutic proteins as described herein in orderto study the effect of therapeutic protein expression in thedisease-susceptible mouse. Transgenic and genetic animal models that aresusceptible to particular disease or physiological conditions, such asstreptozotocin (STZ)-induced diabetic (STZ) mice, are appropriatetargets for expressing variants of FGF19 and/or FGF21, fusions/chimericsequences (or variant) thereof, such as a chimeric peptide sequenceincluding all or a portion of FGF19, or including all or a portion ofFGF21, as set forth herein. Thus, in certain embodiments, there areprovided non-human transgenic animals that produce a variant of FGF19and/or FGF21, or a fusion/chimeric sequence (or variant) thereof, suchas a chimeric peptide sequence including all or a portion of FGF19, orincluding all or a portion of FGF21, the production of which is notnaturally occurring in the animal which is conferred by a transgenepresent in somatic or germ cells of the animal.

The term “transgenic animal” refers to an animal whose somatic or germline cells bear genetic information received, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bymicroinjection or infection with recombinant virus. The term“transgenic” further includes cells or tissues (i.e., “transgenic cell,”“transgenic tissue”) obtained from a transgenic animal geneticallymanipulated as described herein. In the present context, a “transgenicanimal” does not encompass animals produced by classical crossbreedingor in vitro fertilization, but rather denotes animals in which one ormore cells receive a nucleic acid molecule. Transgenic animals providedherein can be either heterozygous or homozygous with respect to thetransgene. Methods for producing transgenic animals, including mice,sheep, pigs and frogs, are well known in the art (see, e.g., U.S. Pat.Nos. 5,721,367, 5,695,977, 5,650,298, and 5,614,396) and, as such, areadditionally included.

Peptide sequences, nucleic acids encoding peptide sequences, vectors andtransformed host cells expressing peptide sequences include isolated andpurified forms. The term “isolated,” when used as a modifier of acomposition provided herein, means that the composition is separated,substantially, completely, or at least in part, from one or morecomponents in an environment. Generally, compositions that exist innature, when isolated, are substantially free of one or more materialswith which they normally associate with in nature, for example, one ormore protein, nucleic acid, lipid, carbohydrate or cell membrane. Theterm “isolated” does not exclude alternative physical forms of thecomposition, such as variants, modifications or derivatized forms,fusions and chimeras, multimers/oligomers, etc., or forms expressed inhost cells. The term “isolated” also does not exclude forms (e.g.,pharmaceutical compositions, combination compositions, etc.) in whichthere are combinations therein, any one of which is produced by the handof man. An “isolated” composition can also be “purified” when free ofsome, a substantial number of, or most or all of one or more othermaterials, such as a contaminant or an undesired substance or material.

As used herein, the term “recombinant,” when used as a modifier ofpeptide sequences, nucleic acids encoding peptide sequences, etc., meansthat the compositions have been manipulated (i.e., engineered) in afashion that generally does not occur in nature (e.g., in vitro). Aparticular example of a recombinant peptide would be where a peptidesequence provided herein is expressed by a cell transfected with anucleic acid encoding the peptide sequence. A particular example of arecombinant nucleic acid would be a nucleic acid (e.g., genomic or cDNA)encoding a peptide sequence cloned into a plasmid, with or without 5′,3′ or intron regions that the gene is normally contiguous within thegenome of the organism. Another example of a recombinant peptide ornucleic acid is a hybrid or fusion sequence, such as a chimeric peptidesequence comprising a portion of FGF19 and a portion of FGF21.

In accordance with the methods provided herein, there are providedcompositions and mixtures of peptide sequences provided herein,including subsequences, variants and modified forms of the exemplifiedpeptide sequences (including the FGF19 and FGF21 variants andsubsequences listed in Table 1 and the Sequence Listing, and theFGF19/FGF21 fusions and chimeras listed in Table 1 and the SequenceListing). In one embodiment, a mixture includes one or more peptidesequences and a pharmaceutically acceptable carrier or excipient. Inanother embodiment, a mixture includes one or more peptide sequences andan adjunct drug or therapeutic agent, such as a bile acid homeostasismodulating or anti-diabetic, or glucose lowering, drug or therapeuticagent. Combinations, such as one or more peptide sequences in apharmaceutically acceptable carrier or excipient, with one or more of abile acid homeostasis modulating or a treatment for a bile acid-relatedor associated disorder, or anti-diabetic, or glucose lowering drug ortherapeutic agent are also provided. Such combinations of a peptidesequence provided herein with another drug or agent, such as a bile acidhomeostasis modulating or acid related disorder treating, or glucoselowering drug or therapeutic agent, for example are useful in accordancewith the methods and uses provided herein, for example, for treatment ofa subject.

Combinations also include incorporation of peptide sequences or nucleicacids provided herein into particles or a polymeric substances, such aspolyesters, carbohydrates, polyamine acids, hydrogel, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers; entrapment in microcapsules prepared by coacervationtechniques or by interfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules, or poly(methylmethacrolate) microcapsules, respectively; incorporation incolloid drug delivery and dispersion systems such as macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems(e.g., N-fatty acyl groups such as N-lauroyl, N-oleoyl, fatty aminessuch as dodecyl amine, oleoyl amine, etc., see U.S. Pat. No. 6,638,513),including oil-in-water emulsions, micelles, mixed micelles, andliposomes, for example.

The peptides provided herein including subsequences, variants andmodified forms of the exemplified peptide sequences (including the FGF19and FGF21 variants and subsequences listed in Table 1 and the SequenceListing, and the FGF19/FGF21 fusions and chimeras listed in Table 1 andthe Sequence Listing) as set forth herein can be used to modulateglucose metabolism and facilitate transport of glucose from the blood tokey metabolic organs such as muscle, liver and fat. Such peptidesequences can be produced in amounts sufficient or effective to restoreglucose tolerance and/or to improve or provide normal glucosehomeostasis.

5.9 Kits

Also provided herein are kits including, but not limited to, peptidesequences provided herein and/or one or more additional agents for thetreatment of a disease, disorder or condition, or a compositioncomprising the foregoing, and one or more pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients, optionallyin further combination with one or more therapeutic agents distinct fromthose described above, compositions and pharmaceutical compositionsthereof, packaged into suitable packaging material. A kit may include alabel or packaging insert including a description of the components orinstructions for use in vitro, in vivo, or ex vivo, of the componentstherein. Exemplary instructions include instructions for treatmentand/or prevention of a disease or disorder. In one embodiment, thedisease or disorder is a bile acid-related or associated disorderprovided herein. In another embodiment, the disease or disorder is ametabolic disorder or associated condition. In yet another embodiment,the disease or disorder is a cancer or tumor.

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.).

Kits provided herein can include labels or inserts. Labels or insertsinclude “printed matter,” e.g., paper or cardboard, separate or affixedto a component, a kit or packing material (e.g., a box), or attached to,for example, an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., hard disk, card, memory disk), optical disk such as CD- orDVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage mediasuch as RAM and ROM or hybrids of these such as magnetic/optical storagemedia, FLASH media or memory type cards. In some embodiments, theinstructions recite a method provided herein.

Labels or inserts can include, among other things, identifyinginformation of one or more components therein, dosing parameters, and/orinformation on the clinical pharmacology of the active ingredient(s),including mechanism of action, pharmacokinetics and pharmacodynamics.Labels or inserts can include information identifying manufacturerinformation, lot numbers, manufacturer location and date.

Labels or inserts can include information on a condition, disorder,disease or symptom for which a kit component may be used. Labels orinserts can include instructions for the clinician or for a subject forusing one or more of the kit components in a method, treatment protocolor therapeutic regimen. Instructions can include dosage amounts,frequency or duration, and instructions for practicing any of themethods, treatment protocols or therapeutic regimens set forth herein.Exemplary instructions include instructions for treatment or use of apeptide sequence as set forth herein and/or the use of an additionalagent or treatment modality useful in treating a bile acid-related orassociated disorder, or a disorder of bile acid homeostasis. Kitsprovided herein therefore can additionally include labels orinstructions for practicing any of the methods and uses provided herein,including treatment methods and uses.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, such as warnings to the subject or clinician regardingsituations where it would not be appropriate to use a particularcomposition. Adverse effects could also occur when the subject has, willbe, or is currently taking one or more other medications that may beincompatible with the composition, or the subject has, will be, or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the composition and, therefore,instructions could include information regarding such incompatibilities.

Kits provided herein can additionally include other components. Eachcomponent of the kit can be enclosed within an individual container andall of the various containers can be within a single package. In certainembodiments, kits are designed for cold storage. Kits provided hereincan further be designed to contain peptide sequences provided herein, orthat contain nucleic acids encoding peptide sequences. Kits providedherein can also be designed to contain, either separately or incombination with the peptide sequences provided herein, one or moreadditional agents useful in the treatment or prevention of a disease ordisorder provided herein. Any cells in the kit can be maintained underappropriate storage conditions until ready to use.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed herein.

In case of conflict, the specification, including definitions, willcontrol. As used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a peptide sequence”or “a treatment,” includes a plurality of such sequences, treatments,and so forth. It is further noted that the claims can be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology such as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges can independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

As used herein, numerical values are often presented in a range formatthroughout this document. The use of a range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention unless the context clearlyindicates otherwise. Accordingly, the use of a range expressly includesall possible subranges, all individual numerical values within thatrange, and all numerical values or numerical ranges including integerswithin such ranges and fractions of the values or the integers withinranges, unless the context clearly indicates otherwise. Thisconstruction applies regardless of the breadth of the range and in allcontexts throughout this patent document. Thus, for example, referenceto a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%,91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of90-100% also includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as wellas 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%,92.5%, etc., and so forth. In addition, reference to a range of 1-3,3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170,170-180, 180-190, 190-200, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a furtherexample, reference to a range of 25-250, 250-500, 500-1000, 1000-2500,2500-5000, 5000-25,000, or 5000-50,000 includes any numerical value orrange within or encompassing such values, e.g., 25, 26, 27, 28, 29 . . .250, 251, 252, 253, 254 . . . 500, 501, 502, 503, 504 . . . , etc. Theuse of a series of ranges includes combinations of the upper and lowerranges to provide another range. This construction applies regardless ofthe breadth of the range and in all contexts throughout this patentdocument. Thus, for example, reference to a series of ranges such as5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includesranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30,10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100,20-150, and so forth.

For the sake of conciseness, certain abbreviations are used herein. Oneexample is the single letter abbreviation to represent amino acidresidues. The amino acids and their corresponding three letter andsingle letter abbreviations are as follows:

alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp(D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly(G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K)methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S)threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V)

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed herein.

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference in its entirety as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedcan be different from the actual publication dates which can need to beindependently confirmed.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the descriptions in the Experimental section are intendedto illustrate but not limit the scope of invention described in theclaims.

6. EXPERIMENTAL

The following is a description of various methods and materials used inthe studies, and are put forth so as to provide those of ordinary skillin the art with a complete disclosure and description of how to make anduse the present invention, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below were performed and are all of theexperiments that may be performed. It is to be understood that exemplarydescriptions written in the present tense were not necessarilyperformed, but rather that the descriptions can be performed to generatethe data and the like associated with the teachings of the presentinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.), but some experimentalerrors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s); s orsec=second(s); min=minute(s); h or hr=hour(s); aa=amino acid(s);kb=kilobase(s); nt=nucleotide(s); pg=picogram; ng=nanogram;μg=microgram; mg=milligram; g=gram; kg=kilogram; pl or pL=picoliter(s);dl or dL=deciliter; μl or μL=microliter; ml or mL=milliliter; 1 orL=liter; μM=micromolar; mM=millimolar; M=molar; kDa=kilodalton;i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC orSQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; TIW=threetimes a week; QM=monthly; HPLC=high performance liquid chromatography;BW=body weight; U=unit; ns=not statistically significant;PBS=phosphate-buffered saline; PCR=polymerase chain reaction;NHS=N-Hydroxysuccinimide; HSA=human serum albumin; BSA=bovine serumalbumin; DMEM=Dulbeco's Modification of Eagle's Medium; GC=genome copy;EDTA=ethylenediaminetetraacetic acid.

6.1 Example 1—Liquid Formulation of M70

Provided below is a liquid formulation design.

Water, Hydrochloric Onmi- Tromethamine Trehalose WFI Acid, Free pHOsmolality Component USP dihydrate quality N.F. TWEEN-20 (25° C.)(Osmol/L) Vendor J T Baker, Pfanstie Corning J T Baker, EMD Avantor hlInc. Avantor Catalog No. 4102-01 6138-23-4 25-055-CM 9544-02 9480 GMPYes Yes No No multicompendial Target amount 2.423 g 92.55 g Add to 0.94mL 0.1 mL 8.000 300 per 1000 mL 999 mL solution Target 20 mM 244.8 mMmolarity Trial 1 2.47 g 92.48 g Add to 0.97 mL 0.1 mL 8.023 299 999 mLTrial 2 2.43 g 92.84 g Add to 0.94 mL 0.1 mL 7.95 303 999 mL Trial 32.42 g 92.43 g Add to 0.94 mL 0.1 mL 7.983 301 999 mL

6.2 Example 2—Stability/Formulation Study Design

All stability studies were performed with protein concentrations of 1mg/mL and 10 mg/ml with the osmolity of 300 Osmol/L for the duration of0 to 24 weeks.

The volume in vials was 0.5-1 mL/vial at temperatures −80° C.; 2-8° C.,and 37° C. at time points of 0 weeks, 1 week, 2 weeks, 4 weeks, 3 monthsand 6 months.

M70 was concentrated to 10+ mg/mL based on A₂₈₀ (MW=21320, E=16180).Concentrated stock was diluted to 1 mg/mL or 10 mg/mL with 1×PBS, andTWEEN-20 (0.01% v/v) was added prior to dialysis step.

Spec pH Actual pH Buffer Composition (4 C.) (4 C.) 1 10 mM Kphos, 150 mMNaCl, 7.3 7.252 0.01% TWEEN-20 2 20 mM Kphos, 125 mM NaCl, 7.3 7.2610.01% TWEEN-20 3 20 mM Kphos, 125 mM NaCl, 7.5 7.540 0.01% TWEEN-20 4 20mM Kphos, 125 mM NaCl, 8.0 8.120 0.01% TWEEN-20 5 20 mM Tris, 8.2%Trehalose, 7.5 7.586 0.01% TWEEN-20 6 20 mM Tris, 8.2% Trehalose, 88.053 0.01% TWEEN-20 7 20 mM Tris, 8.2% Trehalose, 8.5 8.562 0.01%TWEEN-20 8 20 mM Tris, 140 mM NaCl, 7.5 7.499 0.01% TWEEN-20 9 20 mMTris, 140 mM NaCl, 8 8.075 0.01% TWEEN-20 10 20mM Tris, 140 mM NaCl, 8.58.507 0.01% TWEEN-20

Trehalose used for these studies was Cat. No. BP26871 (Fisher). A TrispH temperature dependence was also noted:

5° C. 25° C. 37° C. pH 7.50 6.92 6.72 pH 8.00 7.42 7.15 pH 8.50 7.927.64

The samples were dialyzed overnight into each buffer usingSlide-A-Lyzer® cassette with 10 KMWCO. All vials were flushed with airto remove dust, and then autoclaved on dry cycle for 2 hours at 121° C.to ensure sterility. Samples were sterile filtered through 0.22 μMfilter in fumehood. The vials fills were performed in tissue culturefumehood to ensure stability.

In total, 60 conditions per time point were tested. Vial numbering areused throughout the examples provided herein.

Vial # Buffer Temp Concentration Inject volume 1 1 −80° C. 1 mg/mL 20 21 4° C. 1 mg/mL 20 3 1 37° C. 1 mg/mL 20 4 1 −80° C. 10 mg/mL 2 5 1 4°C. 10 mg/mL 2 6 1 37° C. 10 mg/mL 2 7 2 −80° C. 1 mg/mL 20 8 2 4° C. 1mg/mL 20 9 2 37° C. 1 mg/mL 20 10 2 −80° C. 10 mg/mL 2 11 2 4° C. 10mg/mL 2 12 2 37° C. 10 mg/mL 2 13 3 −80° C. 1 mg/mL 20 14 3 4° C. 1mg/mL 20 15 3 37° C. 1 mg/mL 20 16 3 −80° C. 10 mg/mL 2 17 3 4° C. 10mg/mL 2 18 3 37° C. 10 mg/mL 2 19 4 −80° C. 1 mg/mL 20 20 4 4° C. 1mg/mL 20 21 4 37° C. 1 mg/mL 20 22 4 −80° C. 10 mg/mL 2 23 4 4° C. 10mg/mL 2 24 4 37° C. 10 mg/mL 2 25 5 −80° C. 1 mg/mL 20 26 5 4° C. 1mg/mL 20 27 5 37° C. 1 mg/mL 20 28 5 −80° C. 10 mg/mL 2 29 5 4° C. 10mg/mL 2 30 5 37° C. 10 mg/mL 2 31 6 −80° C. 1 mg/mL 20 32 6 4° C. 1mg/mL 20 33 6 37° C. 1 mg/mL 20 34 6 −80° C. 10 mg/mL 2 35 6 4° C. 10mg/mL 2 36 6 37° C. 10 mg/mL 2 37 7 −80° C. 1 mg/mL 20 38 7 4° C. 1mg/mL 20 39 7 37° C. 1 mg/mL 20 40 7 −80° C. 10 mg/mL 2 41 7 4° C. 10mg/mL 2 42 7 37° C. 10 mg/mL 2 43 8 −80° C. 1 mg/mL 20 44 8 4° C. 1mg/mL 20 45 8 37° C. 1 mg/mL 20 46 8 −80° C. 10 mg/mL 2 47 8 4° C. 10mg/mL 2 48 8 37° C. 10 mg/mL 2 49 9 −80° C. 1 mg/mL 20 50 9 4° C. 1mg/mL 20 51 9 37° C. 1 mg/mL 20 52 9 −80° C. 10 mg/mL 2 53 9 4° C. 10mg/mL 2 54 9 37° C. 10 mg/mL 2 55 10 −80° C. 1 mg/mL 20 56 10 4° C. 1mg/mL 20 57 10 37° C. 1 mg/mL 20 58 10 −80° C. 10 mg/mL 2 59 10 4° C. 10mg/mL 2 60 10 37° C. 10 mg/mL 2

The osometer values of buffers, with or without the presence of M70 wasalso determined on an Advanced Instruments 3300 machine, with 20 μLnominal injection volume:

Buffer 1 mg/mL 10 mg/mL without M70 in M70 in protein buffer bufferBuffer Composition (mOsm) (mOsm) (mOsm) Standard  100 mmol/kg 98/97 — —Standard  290 mmol/kg 284/284 — — Standard 1000 mmol/kg 987/986 — — 1 10mM Kphos, 150 mM 314 316 314 NaCl, 0.01% TWEEN-20 2 20 mM Kphos, 125 mM284 287 293 NaCl, 0.01% TWEEN-20 3 20 mM Kphos, 125 mM 285 288 289 NaCl,0.01% TWEEN-20 4 20 mM Kphos, 125 mM 258 253 254 NaCl, 0.01% TWEEN-20 520 mM Tris, 8.2% 352 360 362 Trehalose, 0.01% TWEEN-20 6 20 mM Tris,8.2% 350 351 350 Trehalose, 0.01% TWEEN-20 7 20 mM Tris, 8.2% 348 342349 Trehalose, 0.01% TWEEN-20 8 20 mM Tris, 140 mM 302 301 303 NaCl,0.01% TWEEN-20 9 20 mM Tris, 140 mM 297 302 302 NaCl, 0.01% TWEEN-20 1020 mM Tris, 140 mM 166 162 160 NaCl, 0.01% TWEEN-20

Visual turbidity assessment was then performed on all the samples at 0,1, 2, 4 weeks. Samples were vortexed to re-suspend any precipitate atthe bottom of the tube. The scoring system used was as follows: 0—Clear;1—Slightly Hazy; 2—Slightly Cloudy; 3—Cloudy; 4—Very Cloudy; and5—Extremely cloudy.

A₃₄₀/A₂₈₀ measurement was performed at 4 weeks (Nanodrop; 1 mmpathlength), and each sample was blanked against respective formulationbuffer.

Analysis for visual turbidity was performed for samples at 37° C. at 4weeks. Results regarding visual observation of samples with the proteinconcentration of 1 mg/mL is depicted in FIG. 1. Results regarding visualobservation of samples with the protein concentration of 10 mg/mL isdepicted in FIG. 2.

The studies showed no evidence of visual precipitate or aggregate forany condition in the samples at −80° C. and 4° C. and at any time point.Variable aggregation was recorded for samples at 37° C. (data notshown), which was pH and protein dose-dependent.

Trehalose was noted to be a superior tonicity modifier over NaCl inTris-containing buffers. Indeed, significant differences in aggregationstate at 37° C. between 20 mM Tris (pH 8.5) with 8.2% trehalose (buffer7) or 140 mM NaCl (buffer 10) were observed.

Buffer 7 (20 mM Tris, 8.2% Trehalose, 0.01% TWEEN-20) was the onlyformulation buffer without evidence of visual precipitates an anycondition, concentration, or time point, and thus was chosen for use infurther studies.

6.3 Example 3—SEC-HPLC Analysis

HPLC analysis was performed on samples with no evidence of precipitationbased on A₂₈₀/A₃₄₀ (ratio ratio≤0.07) and visual inspection (no visualprecipitate) under the following conditions:

-   -   Hardware—HPLC 1200 series    -   Mobile phase—1×PBS in 5% ethanol (v/v)    -   Flow rate—1 mL/min    -   Sample run time—15 min/run    -   Column—Tosoh TSKgel3000    -   Temperature—25° C.    -   Injection volume(s)—20 μL for 1 mg/mL sample (20 μg load); or        -   2 μL for 10 mg/mL sample (20 μg load).

A PBS blank run was performed between every injection to ensure minimalsample carry-over on subsequence injections. A percent (%) recoverybased on time=0 total AUC for each time point (t=1, 2 and 4 weeks) wasalso included.

The data for HPLC analysis based on SEC AUC of absorbance at A₂₁₀ for 1mg/mL in various buffers is shown in FIG. 3; and FIG. 4 shows the datafor HPLC analysis based on SEC AUC of absorbance at A₂₁₀ for Buffer 7 at1 mg/mL. The same data is shown for 10 mg/mL samples in FIG. 5 and FIG.6, respectively.

The results demonstrated that percent (%) recovery based on total AUCwas higher than 92% for all analyzed samples, and which was subsequentlyconfirmed by orthogonal method (AUC_(SV)) (data not shown). Buffer 7also demonstrated a time-dependent increase in percent dimer/aggregateformation at 37° C. The overall trends suggest that the higher the pH,the higher the propensity for dimer and MW aggregates over time inliquid formulation. Moreover, increasing the concentration of M70 onlyhad a minimal effect on the propensity for dimer and higher MWaggregates over time at 4° C. in liquid formulation.

6.4 Example 4—SDS-PAGE Gel Analysis

SDS-PAGE gel analysis was performed on samples with no evidence ofprecipitation based on A₂₈₀/A₃₄₀ and visual inspection (buffers 1-10)under the following conditions:

-   -   Hardware—Novex 4-20% Tris-Glycine gel    -   Sample treatment buffer—Lamelli 3× running buffer ±100 mM DTT.

Samples were diluted to 0.1 μg/μl in 1× Lamelli running buffer. Sampleswere centrifuged at 95° C. for 5 min., and then loaded at 10 μL/lane (1μg total) on the gel. A t=0 reference standard was also used. Aftercompletion, the gel was fixed in 1% acetic acid (v/v) for 30 min.,washed in Milli-Q™ water for 15 minutes, stained in coomassie biosafestain for 1 hour, and then destained overnight in Milli-Q™ water.

The SDS-PAGE gels for Buffer 7 (left) and Buffer 8 (right), for both 1mg/ml and 10 mg/ml are shown in FIG. 7A and FIG. 7B, respectively (otherbuffers; data not shown). The results showed that minimal degradation ofmain M70 peak intensity was observed for all samples tested. Buffer 7contains a non-disulfide dimer band that does not break down uponreduction, with approximately 5% of total protein lane intensity at 4weeks, 37° C. and 10 mg/ml. For the 10 mg/ml sample at 4° C. at 4 weeks,there was no significant increase over the t=0 reference standard forthe dimer band. Moreover, there was no evidence of high molecular weightaggregates in the lane wells for any sample that was tested.

Based on the data from this and the prior experiments, the top 3 bufferswere chosen for further analysis. Buffer rank orders were based on thefollowing analysis at t=0, 1, 2 and 4 week time analysis points:

-   -   Visual inspection (Example 2)    -   A₃₄₀/A₂₈₀ turbity (Example 2)    -   SEC-HPLC (Example 3)    -   SDS-PAGE Gel (Example 4)

With respect to the criteria for selection, the focus was on the 10mg/mL sample analysis to de-risk high concentration formulation (up to10 mg/mL) for clinical needs. An additional focus was on the 4° C.samples, which could provide a base for liquid formulation storageconditions.

In view of the above criteria, Buffers 1, 7 and 10 were chosen forfurther analysis. Buffer 1 had 100% recovery for the 10 mg/mL sample (4weeks a 4° C.), and 98.9% monomer at time point analysis. Moreover,there was minimal non-disulfide covalent dimer at t=4 wk using othermethods for analysis, and showed long-term 4° C. stability (data notshown). Buffer 7 had no precipitation at 37° C. at 10 mg/mL, at t=4 wk;and minimal degradation pathways were observed for 10 mg/mL for 4 weeksat 4° C. (98.9% monomer). Buffer 10 was essentially equivalent to Buffer7; however with NaCl formulation for tonicity (data not shown), andadded diversity for the excipient range.

6.5 Example 5—Ion Exchange Chromatograph-High Performance LiquidChromatography (IEC-HPLC) Analysis

IEC-HPLC analysis of Buffer 7 was performed under the followingconditions:

-   -   Hardware: HPLC 1200 series    -   Mobile phase: (A) 20 mM Tris HCL; pH 8.5; 20% acetonitrile        -   (B) 20 mM Tris HCL; pH 8.5; 20% acetonitrile; 500 mM NaCl    -   Flow rate: 0.5 mL/min with increasing linear gradient of % B    -   Column: Tosoh TSKgel Q-5PW anion exchange column, 7.5 mm×75 mm        (PN 18257)    -   Injection volume: 20 μL for 1 mg/mL sample        -   2 μL for 10 mg/mL sample    -   Time point analysis of samples: Buffer 7 at 1 mg/mL and 10 mg/mL        at t=0, 1, 2 and 4 weeks; Reference standard vs. 37° C.        heat-stressed samples at t=0, 1, 2 and 4 weeks.

FIG. 8 depicts IEC-HPLC for Buffer 7 at 37° C., a concentration of 1mg/mL at t=0 weeks, 1 week, 2 weeks, and 4 weeks. FIG. 9 depictsIEC-HPLC for Buffer 7 at 37° C., a concentration of 10 mg/mL at t=0weeks, 1 week, 2 weeks, and 4 weeks.

6.6 Example 5—Reversed Phase High Performance Liquid Chromatography(RP-HPLC) Analysis

RP-HPLC analysis was performed on samples with no evidence ofprecipitation based on A₃₄₀/A₂₈₀ and visual inspection under thefollowing conditions:

-   -   Hardware: HPLC 1200 series    -   Mobile phase: (A) 0.1 TFA        -   (B) 0.1 TFA, 90% CAN    -   Flow rate: 0.4 mL/min    -   Column Phenomenox™ C4-Jupiter column    -   Injection volume: 20 μL for 1 mg/mL sample        -   2 μL for 10 mg/mL sample    -   Time-point analysis of samples: Buffer 7 at 1 mg/mL and 10 mg/mL        at t=0, 1, 2 and 4 weeks; Reference standard vs. 37° C.        heat-stressed samples at t=0, 1, 2 and 4 weeks.

FIG. 10 depicts RP-HPLC for Buffer 7 at 37° C., a concentration of 1mg/mL at t=0 weeks, 1 week, 2 weeks, and 4 weeks. FIG. 11 depictsRP-HPLC for Buffer 7 at 37° C., a concentration of 10 mg/mL at t=0weeks, 1 week, 2 weeks, and 4 weeks.

6.7 Example 7—Freeze-Thaw Analysis

This analysis was conducted to analyze the effect on M70 formulations ofmultiple freeze/thaw cycles.

M70 was provided at a concentration of 10 mg/mL, and the Osmol/L targetwas 300 Osmol/L. The following buffer composition (similar to Buffer 7)was used: 20 mM Tris pH 8.0 (25° C.), 8.3% (w/v) trehalose, 0.01%TWEEN-20. The fill volume in vials was 1.0 mL/vial, and 6 aliquots werehoused in glass vials that were used in the formulations stabilitystudies discussed above.

The following freeze/thaw conditions were used:

-   -   Number of freeze/thaw cycles: 1, 3, 5    -   Freeze conditions: Place in −80 C for 30 mins.    -   Thaw conditions: (A) Room temperature until thaw (1 hour)        -   (B) 4° C. until thawed (1 hour)

Stability was then assessed using one or more of RP-HPLC, SEC-HPLC orAIEX-HPLC.

FIG. 12 shows the stability of the M70 formulation after the multiplefreeze/thaw cycles. Freeze/thaw cycles do not impact the productstability in the buffer. M70 in 20 mM Tris pH 8.0 (25 C), 8.3% Trehalose(w/v), 0.01% Polysorbate-20 (“TTP” buffer) is stabile during freeze/thawcycling from −80 C to room temperature. There is no detectabledifferences vs. input (ref. std.) for any of the sample conditions oranalyses. Moreover, there is no evidence of reversible oligomerformation via SEC-HPLC using TTP as the mobile phase.

7. SEQUENCE LISTING

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing. The CRF entitled13370-018-228_SEQLIST.txt, which was created on Oct. 14, 2015 and is255,604 bytes in size, is identical to the paper copy of the SequenceListing and is incorporated herein by reference in its entirety.

What is claimed is:
 1. A pharmaceutical composition comprising: a. apeptide having an amino acid sequence comprising or consisting of SEQ IDNO:70 at a concentration of from 1 to 10 mg/mL, and b. apharmaceutically acceptable carrier comprising: i. Tris that is presentin the range of 5 and 50 mM, ii. trehalose that is present in the rangeof between 1 and 20% (w/v), and iii. polysorbate-20 that is present inthe range of from 0.001 to 0.1% (v/v).
 2. The pharmaceutical compositionof claim 1, wherein the peptide has an amino acid sequence comprisingSEQ ID NO:70.
 3. The pharmaceutical composition of claim 1, wherein thepeptide has an amino acid sequence consisting of SEQ ID NO:70.
 4. Thepharmaceutical composition of claim 1, wherein the peptide is at aconcentration of 1 mg/mL.
 5. The pharmaceutical composition of claim 1,wherein the peptide is at a concentration of 5 mg/mL.
 6. Thepharmaceutical composition of claim 1, wherein the peptide is at aconcentration of 10 mg/mL.
 7. The pharmaceutical composition of claim 1,wherein the peptide is fused with an immunoglobulin Fc region.
 8. Thepharmaceutical composition of claim 1, wherein the Tris is present inthe range of 15 and 40 mM.
 9. The pharmaceutical composition of claim 1,wherein the Tris is present in the range of 20 and 35 mM.
 10. Thepharmaceutical composition of claim 1, wherein the Tris is present inthe range of 20 and 30 mM.
 11. The pharmaceutical composition of claim1, wherein the Tris is present in the range of 20 and 25 mM.
 12. Thepharmaceutical composition of claim 1, wherein the Tris is present at 20mM.
 13. The pharmaceutical composition of claim 1, wherein the trehaloseis present in the range of 2 and 15%.
 14. The pharmaceutical compositionof claim 1, wherein the trehalose is present in the range of 3 and 10%.15. The pharmaceutical composition of claim 1, wherein the trehalose ispresent in the range of 4 and 9.5%.
 16. The pharmaceutical compositionof claim 1, wherein the trehalose is present in the range of 5 and9.25%.
 17. The pharmaceutical composition of claim 1, wherein thetrehalose is present in the range of 6 and 9%.
 18. The pharmaceuticalcomposition of claim 1, wherein the trehalose is present in the range of7 and 8.5%.
 19. The pharmaceutical composition of claim 1, wherein thetrehalose is present in the range of 8 and 8.4%.
 20. The pharmaceuticalcomposition of claim 1, wherein the trehalose is present at 8.2%. 21.The pharmaceutical composition of claim 1, wherein the trehalose ispresent at 8.3%.
 22. The pharmaceutical composition of claim 1, whereinthe trehalose is present at 8.4%.
 23. The pharmaceutical composition ofclaim 1, wherein the trehalose is present in the range of 9.2% and 9.3%.24. The pharmaceutical composition of claim 1, wherein the trehalose ispresent at 9.3%.
 25. The pharmaceutical composition of claim 1, whereinthe trehalose is present in the range of 9.24% and 9.25%.
 26. Thepharmaceutical composition of claim 1, wherein the concentration of thepolysorbate-20 is in the range of from 0.0025 to 0.075% (v/v).
 27. Thepharmaceutical composition of claim 1, wherein the concentration of thepolysorbate-20 is in the range of from 0.005 to 0.05% (v/v).
 28. Thepharmaceutical composition of claim 1, wherein the concentration of thepolysorbate-20 is in the range of from 0.0075 to 0.025% (v/v).
 29. Thepharmaceutical composition of claim 1, wherein the concentration of thepolysorbate-20 is 0.01% (v/v).
 30. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition has a pH in a range of7.0 and 9.0 at 4° C. or 25° C.
 31. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition has a pH in a range of7.5 and 8.5 at 4° C. or 25° C.
 32. The pharmaceutical composition ofclaim 1, wherein the pharmaceutical composition has a pH of 8.0 at 4° C.33. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition has a pH of 8.0 at 25° C.
 34. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition does not comprise a salt.
 35. The pharmaceutical compositionof claim 1, wherein the pharmaceutical composition is in a liquid form.36. The pharmaceutical composition of claim 1, wherein less than 10% ofthe peptide aggregates after a period of time when stored at 4° C. or37° C.
 37. The pharmaceutical composition of claim 36, wherein theperiod of time is 4 weeks at 4° C.
 38. The pharmaceutical composition ofclaim 36, wherein the period of time is 4 weeks at 37° C.
 39. Thepharmaceutical composition of claim 1, wherein less than 5% of thepeptide aggregates after a period of time when stored at 4° C. or 37° C.40. The pharmaceutical composition of claim 39, wherein the period oftime is 4 weeks at 4° C.
 41. The pharmaceutical composition of claim 39,wherein the period of time is 4 weeks at 37° C.
 42. The pharmaceuticalcomposition of claim 1, wherein less than 2% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 43. Thepharmaceutical composition of claim 42, wherein the period of time is 4weeks at 4° C.
 44. The pharmaceutical composition of claim 42, whereinthe period of time is 4 weeks at 37° C.
 45. The pharmaceuticalcomposition of claim 1, wherein the composition is provided in asingle-use container.
 46. The pharmaceutical composition of claim 1,wherein the composition is provided in a syringe or an autoinjector. 47.A pharmaceutical composition comprising: a. a peptide having an aminoacid sequence consisting of SEQ ID NO:70 at a concentration of from 1 to10 mg/mL, and b. a pharmaceutically acceptable carrier comprising: i. 20mM Tris, ii. from 8.2% to 8.4% (w/v) trehalose, and iii. 0.01% (v/v)polysorbate-20; wherein the pharmaceutical composition has a pH of 8.0at 25° C.
 48. The pharmaceutical composition of claim 47, wherein thepeptide is present at a concentration of 1 mg/mL.
 49. The pharmaceuticalcomposition of claim 47, wherein the peptide is present at aconcentration of 5 mg/mL.
 50. The pharmaceutical composition of claim47, wherein the peptide is present at a concentration of 10 mg/mL. 51.The pharmaceutical composition of claim 47, wherein the peptide is fusedwith an immunoglobulin Fc region.
 52. The pharmaceutical composition ofclaim 47, wherein the pharmaceutical composition does not comprise asalt.
 53. The pharmaceutical composition of claim 47, wherein thepharmaceutical composition is in a liquid form.
 54. The pharmaceuticalcomposition of claim 47, wherein less than 10% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 55. Thepharmaceutical composition of claim 54, wherein the period of time is 4weeks at 4° C.
 56. The pharmaceutical composition of claim 54, whereinthe period of time is 4 weeks at 37° C.
 57. The pharmaceuticalcomposition of claim 47, wherein less than 5% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 58. Thepharmaceutical composition of claim 57, wherein the period of time is 4weeks at 4° C.
 59. The pharmaceutical composition of claim 57, whereinthe period of time is 4 weeks at 37° C.
 60. The pharmaceuticalcomposition of claim 47, wherein less than 2% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 61. Thepharmaceutical composition of claim 60, wherein the period of time is 4weeks at 4° C.
 62. The pharmaceutical composition of claim 60, whereinthe period of time is 4 weeks at 37° C.
 63. The pharmaceuticalcomposition of claim 47, wherein the composition is provided in asingle-use container.
 64. The pharmaceutical composition of claim 47,wherein the composition is provided in a syringe or an autoinjector. 65.A pharmaceutical composition comprising: a. a peptide having an aminoacid sequence consisting of SEQ ID NO:70 at a concentration of from 1 to10 mg/mL, and b. a pharmaceutically acceptable carrier comprising: i. 20mM Tris, ii. from 9.2% to 9.3% (w/v) trehalose, and iii. 0.01% (v/v)polysorbate-20; wherein the pharmaceutical composition has a pH of 8.0at 25° C.
 66. The pharmaceutical composition of claim 65, wherein thepeptide is present at a concentration of 1 mg/mL.
 67. The pharmaceuticalcomposition of claim 65, wherein the peptide is present at aconcentration of 5 mg/mL.
 68. The pharmaceutical composition of claim65, wherein the peptide is present at a concentration of 10 mg/mL. 69.The pharmaceutical composition of claim 65, wherein the peptide is fusedwith an immunoglobulin Fc region.
 70. The pharmaceutical composition ofclaim 65, wherein the pharmaceutical composition does not comprise asalt.
 71. The pharmaceutical composition of claim 65, wherein thepharmaceutical composition is in a liquid form.
 72. The pharmaceuticalcomposition of claim 65, wherein less than 10% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 73. Thepharmaceutical composition of claim 72, wherein the period of time is 4weeks at 4° C.
 74. The pharmaceutical composition of claim 72, whereinthe period of time is 4 weeks at 37° C.
 75. The pharmaceuticalcomposition of claim 65, wherein less than 5% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 76. Thepharmaceutical composition of claim 75, wherein the period of time is 4weeks at 4° C.
 77. The pharmaceutical composition of claim 75, whereinthe period of time is 4 weeks at 37° C.
 78. The pharmaceuticalcomposition of claim 65, wherein less than 2% of the peptide aggregatesafter a period of time when stored at 4° C. or 37° C.
 79. Thepharmaceutical composition of claim 78, wherein the period of time is 4weeks at 4° C.
 80. The pharmaceutical composition of claim 78, whereinthe period of time is 4 weeks at 37° C.
 81. The pharmaceuticalcomposition of claim 65, wherein the composition is provided in asingle-use container.
 82. The pharmaceutical composition of claim 65,wherein the composition is provided in a syringe or an autoinjector.